Effects of daily administration of tetrahydrocannabinol on rat preovulatory follicles. A quantitative electron-microscopic analysis

Using morphometric techniques, we analyzed the effects of delta 9-tetrahydrocannabinol (THC) on the ultrastructure of rat preovulatory follicles. Rats were injected intraperitonally once daily with vehicle alone, or with vehicle plus THC on diestrus (D), metestrus (M) and diestrus (D), or on estrus (E), M and D. All animals were killed on proestrus. Nuclear, mitochondrial, lipid droplet and lysosomal volume density in the membrana granulosa were analyzed. There was no change in nuclear volume, but there were significant THC-induced decreases in mitochondrial and lipid droplet volumes in the peripheral region of the granulosa. There were increases in dense-body volume in all regions of the granulosa from rats given THC either on D alone, or M and D. Dense-body volume returned to control levels in rats injected on E, M and D. It appears that THC can affect follicular ultrastructure and the effects change with the number of injections.     

An Analysis of Ultrastructural Changes during Oosphere-initial Development in Oogonia from Ca2+-sufficient and Ca2+-deficient cultures of Saprolegnia terrestris Cookson ex Seymour

The central vacuole system of oogonia of Saprolegnia terrestrisfrom Ca²⁺-sufficient cultures was fully enlarged prior to theformation of oosphere initials, which did not involve cleavagevesicles. In oogonia with fully-enlarged central vacuole systems,subsidiary vacuoles at the periphery of the system sometimescontained dense bodies, and dense-body profiles were sometimespresent within sections of the central vacuole system itself.As the central vacuole system enlarged, volume densities ofdense-body vesicles, peripheral vacuoles, lipid bodies and thecytoplasmic matrix decreased relative to total oogonial volume(peripheral protoplasm volume plus central vacuole volume),while the volume density of nuclei increased and that of mitochondriaremained constant. Relative to the peripheral protoplasm only,volume densities of dense-body vesicles, lipid bodies and mitochondriaincreased and volume densities of peripheral vacuoles and ofthe cytoplasmic matrix decreased, while the volume density ofnuclei increased during central vacuole enlargement but subsequentlydecreased during formation of oosphere initials. Under conditionsof Ca²⁺ deficiency, the volume densities of mitochondria andof the cytoplasmic matrix were significantly increased, whilethat of lipid bodies was significantly decreased, at early stagesof oogonial development; the volume densities of other organelleswere not significantly altered at any stage. Saprolegnia terrestris, oogonia, development, calcium, ultrastructure, stereology     

Age-dependent variations in mitochondrial and cytosolic antioxidant enzymes and lipid peroxidation in different regions of central nervous system of guinea pigs

The age-related changes in the activities of antioxidant enzymes of mitochondrial and cytosolic fractions were measured in different regions of the central nervous system (CNS) in 10 and 32 months old guinea pigs. In old animals, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were reduced (p < 0.05) in all the regions of CNS studied but catalase (CAT) declined significantly only in the cerebral cortex, hypothalamus and cerebellum. Glutathione reductase (GRd) activity declined in cerebral cortex and hypothalamus in the cytosolic fractions and only in cerebellum in the mitochondrial fraction. It is concluded that age-related decline in the activities of antioxidant enzymes is both region and enzyme specific. The endogenous lipid peroxide was found to be significantly higher (p < 0.05) in the 32 month old animals whereas, lipid peroxidation after incubating the tissue homogenate in air was found to be lower (p < 0.05). The in vitro mitochondrial lipid peroxidation decreased with age. The results indicate that accumulation of lipid peroxides takes place with ageing but the susceptibility of lipid peroxidation decreases in the older animals.     

Environmentally induced changes in mitochondria and endoplasmic reticulum of Saccharomyces carlsbergensis yeast

The effects of culture environment on the volume density and surface density of mitochondria and endoplasmic reticulum in a facultative yeast were studied. When compared with cells grown aerobically on a nonrepressive substrate, cells grown in the absence of oxygen showed a sharp reduction in both volume density of mitochondria and surface density of the inner mitochondrial membrane (imm) in the remaining mitochondrial profiles. Use of fermentable (repressive) substrates under aerobic conditions restricted the volume density of mitochondria to a much greater extent than the surface density of imm. The range of mitochondrial volume densities in these experiments was 4-11%. Surface density of endoplasmic reticulum (ER) was sensitive to growth rate and in particular to changes in oxygen tension, showing large fluctuations during both anaerobic and aerobic adaptation. These fluctuations in ER are discussed in relation to the known role of this organelle in lipid metabolism.     

A QUANTITATIVE DESCRIPTION OF CORTISONE-INDUCED ALTERATIONS IN THE ULTRASTRUCTURE OF RAT LIVER PARENCHYMAL CELLS

A stereological comparison of the hepatic parenchymal cells from 125-g male rats given a daily injection for 6 days of either 5 mg of cortisone acetate or saline (controls) was carried out with both light and electron microscopy. Cortisone treatment results in an increase in average parenchymal cell cytoplasmic volume from 5100 to 5800 µ³ and a decrease in average nuclear diameter from 7.1 to 6.5 µ. The volume of the average mitochondrion is increased fourfold in midzonal and peripheral regions of hepatic lobules, and there is a decrease in the number of mitochondria per cell such that the total mitochondrial volume per cell remains approximately unchanged. The numbers of peroxisomes are reduced, while the numbers of lysosomes and lipid droplets are increased in all parts of the lobules. The average volume of glycogen is doubled in all cells. The areas of membranes of the smooth- and rough-surfaced endoplasmic reticulum are decreased to one-half and two-thirds of their control values, respectively. The effects of cortisone on these various structural elements is discussed with respect to steroid-related alterations in biochemical processes.     

GNRH induces activation of Leydig-like cells in Pleurodeles waltlii. A morphometric study

The ultrastructure of the interstitial cells of the glandular tissue of Pleurodeles waltlii was studied in testis of animals obtained in early breeding season (January) under gonadotropic releasing hormone (GNRH) treatments and controls. These cells (parenchymal or Leydig-like cells) displayed the structural characteristics of steroid-producing cells. GNRH administration for 24 hours induced a significant decrease of both medial volume and volume density of lipid droplets. On the other hand, cell volume, nucleus, mitochondria, mitochondrial cristae and tubules of smooth endoplasmic reticulum were increased. The surface density of mitochondrial cristae was also increased.     

Raman and fluorescence imaging of phospholipidosis induced by cationic amphiphilic drugs in endothelial cells

Cationic amphiphilic drugs (CADs) are known from lysosomotropism, drug-induced phospholipidosis (DIPL), activation of autophagy, and decreased cell viability, but the relationship between these events is not clear and little is known about DIPL in the endothelium. In this work, the effects of fluoxetine, amiodarone, clozapine, and risperidone on human microvascular endothelial cells (HMEC-1) were studied using a combined methodology of label-free Raman imaging and fluorescence staining. Raman spectroscopy was applied to characterize biochemical changes in lipid profile and their distribution in the cellular compartments, while fluorescence staining (LysoTracker, LipidTOX, LC3B, and JC-1) was used to analyze lysosome volume expansion, activation of autophagy, lipid accumulation, and mitochondrial membrane depolarization. We demonstrated that fluoxetine, amiodarone, and clozapine, but not risperidone, at non-toxic concentrations induced lipid accumulations in the perinuclear and cytoplasmic regions of endothelial cells. Spectroscopic markers of DIPL included a robust increase in the ratio (lipid/(protein + lipid)), an increase in choline-containing lipid, fatty acids, and the presence of cholesterol esters, while starvation-induced activated autophagy revealed a spectroscopic signature associated with subtle changes in the lipid profile only. Interestingly, lysosomal volume expansion, occurrence of DIPL, and activation of autophagy induced by selected CADs all depended on drug-accumulation in acidic pH of lysosome cellular compartments whereas reduced endothelial viability did not, and was attributed to mitochondrial mechanisms as evidenced by a decreased mitochondrial transmembrane potential. In conclusion, drug-induced phospholipidosis in the endothelium did not reduce endothelial viability per se and can be efficiently assayed by Raman imaging.     

Stereological study of B-cell mitochondria in alloxan-treated mice.

Using stereological techniques, including semi-automatic image analysis, the B-cell mitochondria were studied in the pancreatic islets from one group of control mice and two groups of mice killed 10 min and 60 min, respectively, after alloxan administration. Ten min following alloxan the mitochondrial volume and envelope surface densities, the mean mitochondrial volume and surface area, and the area of mitochondrial profiles were significantly increased, whereas the mitochondrial numerical density was not significantly altered. At the 60 min observation time the mitochondrial volume density, the mean mitochondrial volume and surface areas, and the area of mitochondrial profiles were significantly decreased, whereas the mitochondrial envelope surface was not significantly altered. The findings indicate a rapid swelling, followed by disintegration of the mitochondria in the B-cells of alloxan-treated mice, thereby supporting our view that mitochondrial lesions play a primary role in the development of alloxan diabetes. These lesions are believed to be due to ionic alterations in the B-cells ("Pi-pH hypothesis").     

Variation of mitochondrial volume fraction along multiply innervated fibers in rabbit extraocular muscle

Mitochondrial volume fraction was compared among three regions along the length of six multiply innervated fibers (MIFs) in the orbital surface layer of rabbit superior rectus. These MIFs are of about 5 μm diameter toward the middle of their length, and of about 15 μm diameter toward their proximal and distal ends. The region of highest volume fraction (26%) was located toward the proximal end of their segment of minimal diameter, in apparent association with endplate-like nerve junctions. The region of lowest volume fraction (8%) was located at their distal segment of maximal diameter. The region toward the distal end of their segment of minimal diameter displayed an intermediate volume fraction (15%). These mitochondrial volume fractions were further analyzed in terms of the relative contributions of the I-band, the A-band, and the subsarcolemmal mitochondrial clusters. Comparable changes in mitochondrial content occur in both the I-band and A-band: in the fibers' distal segment of maximal diameter, however, the mitochondrial volume fraction in the A-band (5%) is lower than in the I-band (11%). These modifications of mitochondrial content along the fibers' length occur irrespective of the contributions of the subsarcolemmal mitochondrial clusters.     

Role of skeletal muscle mitochondrial density on exercise-stimulated lipid oxidation

Reduced skeletal muscle mitochondrial density is proposed to lead to impaired muscle lipid oxidation and increased lipid accumulation in sedentary individuals. We assessed exercise-stimulated lipid oxidation by imposing a prolonged moderate-intensity exercise in men with variable skeletal muscle mitochondrial density as measured by citrate synthase (CS) activity. After a 2-day isoenergetic high-fat diet, lipid oxidation was measured before and during exercise (650 kcal at 50% VO(2)max) in 20 healthy men with either high (HI-CS = 24 ± 1; mean ± s.e.) or low (LO-CS = 17 ± 1 nmol/min/mg protein) muscle CS activity. Vastus lateralis muscle biopsies were obtained before and immediately after exercise. Respiratory exchange data and blood samples were collected at rest and throughout the exercise. HI-CS subjects had higher VO(2)max (50 ± 1 vs. 44 ± 2 ml/kg fat free mass/min; P = 0.01), lower fasting respiratory quotient (RQ) (0.81 ± 0.01 vs. 0.85 ± 0.01; P = 0.04) and higher ex vivo muscle palmitate oxidation (866 ± 168 vs. 482 ± 78 nmol/h/mg muscle; P = 0.05) compared to LO-CS individuals. However, whole-body exercise-stimulated lipid oxidation (20 ± 2 g vs. 19 ± 1 g; P = 0.65) and plasma glucose, lactate, insulin, and catecholamine responses were similar between the two groups. In conclusion, in response to the same energy demand during a moderate prolonged exercise bout, reliance on lipid oxidation was similar in individuals with high and low skeletal muscle mitochondrial density. This data suggests that decreased muscle mitochondrial density may not necessarily impair reliance on lipid oxidation over the course of the day since it was normal under a high-lipid oxidative demand condition. Twenty-four-hour lipid oxidation and its relationship with mitochondrial density need to be assessed.     

Rat soleus muscle ultrastructure after hindlimb suspension

The aim of the present investigation was to determine, by quantitative electron microscopy, the effects of a 5-wk tail-suspension period on rat soleus muscle ultrastructure. A marked decline (-60%) in muscle mass occurred. The mean fiber cross-sectional area decreased to a greater extent (-75%) than the capillary-to-fiber ratio (-37%), leading to a higher capillary density (+148%) after hypokinesia. The total mitochondrial volume density remained unchanged, whereas the volume density of myofibrils was slightly but significantly reduced (-6%). A shift from subsarcolemmal to interfibrillar mitochondria occurred. Interfibrillar mitochondrial volume density was highest near the fiber border and decreased toward the fiber center. An increase in volume density of satellite cells suggested muscle regenerative events. Soleus atrophy with tail suspension greatly decreases the muscular volume but leaves the ultrastructural composition of muscle fibers relatively unaffected.     

Stereological analysis of ultrastructural changes in cardiomyocytes during adriblastina-induced cardiomyopathy

Both single and graded administration of an anthracycline antibiotic adriablastine at a dose of 20 mg/kg to white rats have resulted in cardiomyocyte volume decrease, dystrophia and lysis of myofibrils, and reduction of mitochondrial surface density volume. Accumulations of myelin bodies of different configuration and diffuse myolysis were observed. Myelin bodies and diffuse myolysis of cardiomyocytes in adriablastine-induced cardiomyopathy are believed to be a morphological sign of lipid peroxidation.     

High aerobic capacities in the skeletal muscles of pinnipeds: adaptations to diving hypoxia.

The objective was to assess the aerobic capacity of skeletal muscles in pinnipeds. Samples of swimming and nonswimming muscles were collected from Steller sea lions (Eumetopias jubatus, n = 27), Northern fur seals (Callorhinus ursinus, n = 5), and harbor seals (Phoca vitulina, n = 37) by using a needle biopsy technique. Samples were either immediately fixed in 2% glutaraldehyde or frozen in liquid nitrogen. The volume density of mitochondria, myoglobin concentration, citrate synthase activity, and beta-hydroxyacyl-CoA dehydrogenase was determined for all samples. The swimming muscles of seals had an average total mitochondrial volume density per volume of fiber of 9.7%. The swimming muscles of sea lions and fur seals had average mitochondrial volume densities of 6.2 and 8.8%, respectively. These values were 1.7- to 2.0-fold greater than in the nonswimming muscles. Myoglobin concentration, citrate synthase activity, and beta-hydroxyacyl-CoA dehydrogenase were 1.1- to 2. 3-fold greater in the swimming vs. nonswimming muscles. The swimming muscles of pinnipeds appear to be adapted for aerobic lipid metabolism under the hypoxic conditions that occur during diving.     

UCP-mediated energy depletion in skeletal muscle increases glucose transport despite lipid accumulation and mitochondrial dysfunction

To address the potential role of lipotoxicity and mitochondrial function in insulin resistance, we studied mice with high-level expression of uncoupling protein-1 in skeletal muscle (UCP-H mice). Body weight, body length, and bone mineral density were decreased in UCP-H mice compared with wild-type littermates. Forelimb grip strength and muscle mass were strikingly decreased, whereas muscle triglyceride content was increased fivefold in UCP-H mice. Electron microscopy demonstrated lipid accumulation and large mitochondria with abnormal architecture in UCP-H skeletal muscle. ATP content and key mitochondrial proteins were decreased in UCP-H muscle. Despite mitochondrial dysfunction and increased intramyocellular fat, fasting serum glucose was 22% lower and insulin-stimulated glucose transport 80% higher in UCP-H animals. These beneficial effects on glucose metabolism were associated with increased AMP kinase and hexokinase activities, as well as elevated levels of GLUT4 and myocyte enhancer factor-2 proteins A and D in skeletal muscle. These results suggest that UCP-H mice have a mitochondrial myopathy due to depleted energy stores sufficient to compromise growth and impair muscle function. Enhanced skeletal muscle glucose transport in this setting suggests that excess intramyocellular lipid and mitochondrial dysfunction are not sufficient to cause insulin resistance in mice.     

自发性高血压大鼠脑动脉心钠素受体分布的计量学研究

用软脑膜铺片技术,兔疫组织化学方法及全自动图像分析仪对１７周龄自发性高血压大鼠脑血管床心钠素受体分布密度进行研究、结果表明,自发性高血压大鼠脑小动脉及细动脉壁上心钠素受体分布密度明显低于正常血压大鼠,Ｐ＜０．０１。此外,在细小动脉分叉部位心钠素受体分布密度增加,而自发性高血压大鼠中其分叉部位的受体密度明显低于正常血压大鼠。从实验结果显示,我们试用的软脑膜铺片技术显然弥补了组织切片中从血管横断面观察心钠素受体分布的局限性,清楚地展现了整个血管床心钠素受体分布特征。根据实验结果提示这些阻力性动脉心钠素受体密度减少与细小动脉舒血管反应减弱,外周阻力增加,血压升高有着明显的关系;尤其是对调节外周循环血量和外周阻力充当闸门作用的细小动脉分叉部位,心钠素受体密度降低这一特征在高血压发生机制中可能起着重要作用。     

Comparative studies of the physical state of the lipid phase of normal and hypercholesterolemic very low density lipoprotein.

Very low density lipoproteins (VLDL) were prepared from the serum of rabbits at various stages of hypercholesterolemia (95--1665 mg cholesterol/100 ml of serum). The most notable chemical change in hypercholesterolemic (hc) VLDL was the greatly increased content of cholesteryl esters and the greatly decreased content of triglycerides, compared to normal (n) VLDL. Structurally, the lipid region of n VLDL possessed a much lower microviscosity than did hc VLDL, when analyzed by fluorescence polarization and pyrene eximer methods. The microviscosity of the redispersed n VLDL lipid extract was considerably greater than the observed in n VLDL; but less than that of hc VLDL. Incorporation of pyrene into the lipid region of n VLDL and hc VLDL allowed assessment of various properties of the surface and hydrocarbon regions of these lipoproteins. Only slight differences were found in the pyrene monomer 3 : 1 fluorescence emission peak ratios, and in the rate constant for quenching of pyrene by O2. However, the quenching rate constant of pyrene by I- and iodoheptane were different for each lipoprotein.     

Enhanced hippocampal F2-isoprostane formation following kainate-induced seizures

We attempted to obtain evidence for the occurrence of oxidant injury following seizure activity by measuring hippocampal F2-isoprostanes (F2-IsoPs), a reliable marker of free radical-induced lipid peroxidation. Formation of F2-IsoPs esterified in hippocampal phospholipids was correlated with hippocampal neuronal loss and mitochondrial aconitase inactivation, a marker of superoxide production in the kainate model. F2-IsoPs were measured in microdissected hippocampal CA1, CA3 and dentate gyrus (DG) regions at various times following kainate administration. Kainate produced a large increase in F2-IsoP levels in the highly vulnerable CA3 region 16 h post injection. The CA1 region showed small, but statistically insignificant increases in F2-IsoP levels. Interestingly, the DG, a region resistant to kainate-induced neuronal death also showed marked (2.5-5-fold) increases in F2-IsoP levels 8, 16, and 24 h post injection. The increases in F2-Isop levels in CA3 and DG were accompanied by inactivation of mitochondrial aconitase in these regions. This marked subregion-specific increase in F2-Isop following kainate administration suggests that oxidative lipid damage results from seizure activity and may play an important role in seizure-induced death of vulnerable neurons.     

Cytological effects of platelet-derived growth factor on mitochondrial ultrastructure in fibroblasts

The goal of this study was to evaluate morphofunctional changes in mitochondrial ultrastructure after platelet-derived growth factor application in fibroblasts as an indicator of mitochondrial activation in processes like wound healing. NRK-49F fibroblasts were synchronized, incubated with PDGF (platelet-derived growth factor) and studied by electron microscopy. Volume density (Vv), numerical density (Nv) and surface density (Sv) were measured by stereological analysis. Application of PDGF on NRK-49F caused an increase in mitochondrial volume density by 57% and surface area of cristae per mitochondrion by 65%. The numerical density of the mitochondria was decreased in the PDGF-treated cells by 23%, but at the same time their mean volume was increased. Furthermore, the mitochondria had a complex and highly variable shape both in control and PDGF-treated cells, possibly indicating the existence of a mitochondrial reticulum. The results demonstrated that biochemically active membrane systems in fibroblast mitochondria are enlarged as a direct effect of small doses of platelet-derived growth factor and support the concept that this factor and related peptides serve as mitogens for connective tissue forming cells. Thus, in mitogenic processes like wound healing, the high energy demand of fibroblasts is provided by the increase of the inner surface of mitochondria.     

运动性骨骼肌疲劳亚细胞机制的探讨

本实验采用持续性下坡跑运动,观察大鼠骨骼肌运动后不同时相线粒体形态、代谢、机能等指标的变化,结果表明：大鼠运动后即刻线粒体钙含量、细胞膜丙二醛（ＭＤＡ）值明显增加,ＡＴＰ含量和细胞膜Ｎａ＋,Ｋ＋－ＡＴＰ酶活性下降;运动后２４ｈ线粒体钙含量、ＭＤＡ值增加最明显,ＡＴＰ含量仍未恢复,细胞膜Ｎａ＋,Ｋ＋－ＡＴＰ酶活性基本恢复,线粒体体密度、平均体积比运动前明显增加,比表面缩小;运动后４８ｈＡＴＰ含量完全恢复,线粒体钙含量、ＭＤＡ值开始恢复。本研究结果提示,急性运动引起的细胞膜脂质过氧化加强、线粒体形态、代谢机能异常抑制线粒体氧化磷酸化过程、减少ＡＴＰ生成可能是运动性骨骼肌疲劳的亚细胞机制之一。耐力训练可以通过改善线粒体形态、代谢、机能提高机体的运动能力。     

Thermal compensation of peripheral oxygen transport in skeletal muscle of seasonally acclimatized trout

Seasonal changes in ultrastructure of locomotory muscle were quantified after acclimatization to natural temperature and photoperiod. Only modest changes were seen in the volume density (V(v)) of mitochondria in slow fibers ranging from 0.21 +/- 0.01 (summer) to 0.24 +/- 0.01 (winter), despite an increase in fiber size from 945 +/- 19 to 1,594 +/- 46 microm(2), respectively, resulting in a significantly greater total mitochondrial volume at low temperatures. In contrast, intracellular lipid stores showed a marked change with season, from a maximum V(v) of lipid droplets of 0.16 +/- 0.01 in winter, progressively declining through spring and summer to a minimum of 0.07 +/- 0.01 in autumn. For both organelles, the surface density reflected changes in V(v), indicating little modification of structure. Seasonal effects may dominate those of environmental temperature on mitochondrial separation, which in winter and spring fish at 4(o)C averaged 0.64 +/- 0.06 and 1.20 +/- 0.07 microm, respectively. The extracellular transport of oxygen also varies with season, the peak capillary density in autumn (2,851 +/- 88 mm(-2)) resulting in a minimum tissue supply (domain) area of 529 +/- 9 microm(2) per capillary. As a consequence, the predicted intracellular PO(2) ( approximately 2.5 kPa) is similar throughout the year.