Electron microscopic observations of the conjunctiva in epidermolysis bullosa hereditaria

PURPOSE: Our purpose was to assess the effect of cryopreservation on cytoskeleton of germinal vesicle (GV) mouse oocytes and determine whether irreversible spindle damage and related digyny associated with cryopreservation of metaphase II (MII) oocytes can be avoided. METHODS: The GV oocytes were cryopreserved using a slow-cooling (0.5 degree C/min) and slow-thawing (8 degrees C/min) protocol in 1.5 M dimethylsulfoxide supplemented with 0.2 M sucrose and analyzed before and during fertilization by multiple-label fluorescence and differential interference contrast microscopy techniques. RESULTS: When examined after in vitro maturation, the vast majority (> 95%) of cryopreserved and control oocytes displayed normal microfilament and microtubule organization. With respect to barrel-shaped spindle and normal chromosome alignment, no significant differences were observed between cryopreservation (78 and 86%, respectively) and control (85 and 95%, respectively) groups. In fertilization experiments, spindle rotation, formation of the second polar body, and pronuclear migration were displayed by similar percentages of cryopreserved (96, 94, and 37%, respectively) and control (98, 97, and 45%, respectively) oocytes, indicating normal functionality of the cytoskeleton during this period. However, pronuclear formation was significantly inhibited by cryopreservation (81%) compared with controls (100%). Regarding digyny and polyspermy, no significant increase was observed after cryopreservation (3 and 10%, respectively) compared with controls (3 and 6%, respectively). CONCLUSIONS: Cryopreservation of mouse oocytes at the GV stage is particularly advantageous to circumvent the spindle damage and increased digyny noted after cryopreservation of MII oocytes.     

Electron microscopic observations on the elimination of the oocyte through the peritoneal epithelium in the neonatal mouse ovary

Six girls and one boy with cronic active hepatitis (CAH) of unknown etiology were between 9 and 15 years at the clinical onset of their illness. After beginning immunosuppressive therapy the course of their disease was followed from one to ten years. All had markedly increased IgG, high titres of smooth muscle antibodies (SMA) and antinuclear antibodies of IgG class in the earliest serum specimens tested. Therapy resulted in an improved sense of well-being and a decrease in SGOT, IgG and titres of SMA. Very high titres of measles antibodies were observed in all cases. In one of the cases CAH manifested itself after measles and in another after rubella infection. The first case in our series of patients died of liver failure after 5 years of illness. The other patients have survived and are able to live a normal life. The possibility of CAH should be considered when children develop symptoms of hepatitis. Longterm immunosuppressive treatment with regular clinical and laboratory evaluation is important. Estimation of titres of SMA is an additional parameter of value in following of the activity of CAH in these young patients.     

Electron microscopic observations on the formation of elastic fibers in primary cultures of aortic smooth muscle cells

Increasing experimental concern with locomotion and its neural control prompted the construction of a motor-driven treadmill for cats. The apparatus was designed for kinematic analysis of gait from 16 mm movie film, but it will also permit a variety of other records to be taken. A box, or base, houses two large drums and the belt drive. Space is provided on top of the base for a closed locomotion compartment that is transparent on the filming side. There is also sufficient space on top of the base for support apparatus, operator controls, and animal exits. Cats may be trained to work for food reward alone, as delivered from the front by the operator. Alternatively, they can be trained to stay near the front of the treadmill belt in order to avoid an aversive jet of air or electric shock, either of which can be delivered at the rear of the test chamber. The operator can maintain forward belt velocity at fixed levels between 0.5 and 6 m/sec, or he can vary velocity continuously. Displays of a tachometer and a special timing device are large enough to be clearly visible on the film. These various features in combination provide a controlled and varsatile stepping environment, in which the cat can move without physical restraints.     

Electron microscopic determination of orientation

The martensites of titanium binary alloys, containing 1, 2, and 5.3 wt pct Cu, were studied by transmission electron microscopy techniques. Roughly parallel plates with different variants of the orientation relationship, as well as colonies of identical variant plates, prevail in the structures. A stereographic projection analysis of super-imposed diffraction patterns from two adjacent plates is shown to be sufficient for the deduction of the orientation relationship that existed during transformation, which is found to be the Burgers’ relationship -(110)_β∥ (0001)_α′; 〈111〉_β∥ 〈2110〉_α′. A graphical method was developed for the derivation of the habit plane and its particular variant, in spite of the absence of retained β phase in quenched Ti-Cu alloys. The habit plane of Ti-Cu martensite is found to be (1079)_β with 4 deg accuracy, and to best agree with Class A (α^- ω⁺) solution of the Bowles and Mackenzie theory, rather than with (α⁺ ω⁺) or with Class B solutions.     

Electron microscopic detection of glycoconjugates in the chicken lens

The cytochemical localization of glycoconjugates in the 14-day old embryonic chick lens was analysed by lectin-gold labelling. Con A/HRP gold particles, specific for D-mannose labelled the interior of the rough endoplasmic reticulum, membranes of the Golgi complex, secretory vesicles and the plasma membranes of the lens epithelial cell. The lens capsule was heavily labelled. Lens fiber cell membranes were also labelled. In contrast LFA, specific for neuraminic acid, did not bind to the endoplasmic reticulum or nuclear membrane. Labelling of the Golgi complex, secretory vesicles and capsule was observed. The plasma membranes of epithelial and fiber cells were extensively labelled, and probably reflects the presence of glycolipids such as gangliosides.     

Electron microscopic studies on the aryl-sulphatase activity of the pineal gland

On applying p-Nitrocatechinsulphate (2-Hydroxy-5-nitrophenylsulphate) as substrate, characteristic positivity was demonstrable in the pineal gland. The reaction was localized to the Golgi system and to the smooth endoplasmic reticulum, producing in them a homogenous, in the primary lysosomes a fine granulated strongly electron scattering precipitate. The development of primary lysosomes was well detectable with histochemical reaction at the level of both the Golgi apparate and smooth endoplasmic reticulum. Membrane limited bodies occurring in the cells corresponded to autolysosomes. Negative reaction was obtained with para-nitro-phenylsulphate and acethyl-phenylsulphate. The presence of KCN and pCMB inhibited the reaction.     

Anatomical and neurohistological observations on the heart of the rose ringed parakeet, Psittacula krameri

Anatomy, histology and innervation of the heart of the rose ringed parakeet, Psittacula krameri have been studied in the present investigation. The sinuatrial node is found to be well-developed. It is located towards the right side of the cephalic end of the interatrial septum and composed of a few nucleated cells and a large fibrous mass. The atrioventricular node is poorly defined, present at the caudal end of the interatrial septum. The node is somewhat triangular in shape and is composed of elongated and multinucleated specialized fibres. The node is not covered by any connective tissue sheath. The poor development of the atrio ventricular node and the absence of any sheath around it may be correlated with the fast rate of the heart beat. The atrioventricular bundle is observed at the cephalic end of the interventricular septum. A branch from the right limb of the atrioventricular bundle is noted to pass directly into the right atrioventricular valve. The heart is richly innervated. Ganglion cells along with nerve fibres have been observed at the sulcus terminalis and the atrioventricular junction. A direct nervous connection could be observed between the sinuatrial and atrioventricular nodes. It is argued that the impulse which originates in the sinuatrial node would reach the atrioventricular node through the unspecialized muscle fibres and nerve fibres of the interatrial septum. Nerve cells could not be traced in the substance of the sinuatrial node, atrioventricular node and atrioventricular bundle.     

Specular microscopic observations of the corneal endothelium in the normal rabbit

Endothelial specular microscopy and pachometry were performed on both eyes of 14 young adult New Zealand white rabbits with clinically normal eyes. Endothelial cells of the central corneas formed a mosaic-like pattern of homogenous hexagonal cells with a mean diameter of 20.6 +/- 1.0 micron sd. The mean number of cells per mm was 2998 +/- 326 sd and the mean corneal thickness was 0.38 +/- 0.02 mm sd.     

Electron microscopic and autoradiographic studies on vitellogenesis in Necturus maculosus

Electron microscope studies on Necturus maculosus oocytes ranging in size from 1.1-1.5 mm in diameter indicate the primary proteinaceous yolk to arise within structures referred to in other amphibian oocytes as yolk precursor sacs or bodies. The origin of these yolk precursor sacs appears to result from the activity of the Golgi complexes which form multivesicular and granular-vesicular bodies, the limiting membrane of which is at times incomplete. During differentiation, the yolk precursor sacs contain small vesicles similar in size to Golgi vesicles, larger vesicles similar to vesicular elements of the agranular endoplasmic reticulum and, on occasion, a portion of a mitochondrion. The interior of these sacs becomes granular, perhaps by a dissolution of the components just described, and soon becomes organized into a crystalline configuration. In oocytes 2.0-2.5 mm in diameter, an extensive micropinocytotic activity begins, continues throughout vitellogenesis, and constitutes the primary mechanism for the formation of secondary yolk protein. Numerous coated and smooth-surfaced vesicles, as well as electron-dense and electronlucent ones, fuse in the cortical ooplasm to form progressively larger yolk platelets.     

Electron microscopic observation on a non-occluded baculo-like virus in shrimps

A severe disease of farm-raised shrimp, Penaeus chinensis has spread throughout Chinese coasts since 1993. Recently, a baculo-like virus has been diagnosed as the causative agent for this epidemic disease. The electron microscopic observation of the virus is described. Thin sections of hepatopancreatic and hypodermic tissue of diseased P. chinensis showed many rod-shaped, enveloped, baculo-like virions in hypertrophied nuclei of infected cells. The virion was filled with a highly electron dense core. No occlusion bodies have been found. Negative stained intact virions, purified from infected tissues by centrifugation on sucrose discontinuous gradients, demonstrated that the viral envelops had been broken, but the cylindrical nucleocapside could be observed clearly. The nucleocapsid of average 62 nm x 314 nm was composed of a helix system of capsomers, giving rise to an open stacked ring structure and repeating approximately every third ring. The number of repeating unit was 13 to 15. We propose that the virus described here could be designated as Non-Occluded Shrimp Virus (NOSV) or Penaeus chinensis Baculo-Like Virus (PcBLV).     

The correlation of sperm chromatin decondensation following in vitro exposure to heparin and sperm penetration rates

PURPOSE: The aim of this study was to evaluate the possible correlation of low-dose heparin-induced decondensation of sperm chromatin with sperm concentration, motility, morphology, membrane hypoosmotic response, ejaculate volume, and the ability of sperm to penetrate zona-free hamster oocytes. METHODS: Twenty-two donors of known fertility and 105 patients undergoing evaluation at an andrology laboratory were evaluated by standard World Health Organization semen analysis techniques and a modified sperm penetration assay (SPA). An aliquot was also incubated for 60 min and Ham's F10 medium containing 50 USP/ml heparin. The percentage of sperm undergoing chromatin decondensation was evaluated and correlated to SPA rates and semen quality parameters. RESULTS: No significant correlation was observed between semen parameters and decondensation rates. A nonsignificant (P = 0.11) inverse correlation (P = -0.21) was observed between SPA rates and chromatin decondensation. Significant (P < 0.001) differences were observed in the decondensation rate of donors (3.7 +/- 0.6), patients with normal SPA rates (7.8 +/- 1.5), and patients with decreased SPA rates (21.7 +/- 1.8). The decondensation rates were significantly different (P < 0.01) between patients with a normal SPA rate and patients with a decreased SPA rate. CONCLUSIONS: These data indicate a significant inverse relationship between the SPA rate, which has previously been shown to correlate highly with fertilization ability and heparin-induced sperm chromatin decondensation.     

Hatching of Schistosoma mansoni eggs and observations on motility of miracidia

Eggs of Schistosoma mansoni were obtained from livers of mice and hatching was observed under varying conditions of light and ionic composition of the medium. Hatching occurred equally well in light and in darkness. Eggs also hatched readily in 1- to 50-m OsM solutions of urea, sucrose, sodium chloride, and glycerol, but hatching was inhibited at higher concentrations unless the eggs were left in solutions for long periods of time. Hatching readily occurred in deionized water, but the emerged miracidia did not swim longer than 5 to 10 min unless Na+ was added. Histochemistry of the egg showed DNA-positive egg granules and a polysaccharide-positive vacuole matrix. Acid mucopolysaccharides were stained in the vacuolar matrix and in the anterior sac of the miracidium. Longitudinal alignment of constituents of the egg shell is suggested by the predominance of longitudinal rents in the shell at hatching. A mechanism of hatching involving an osmotic stimulus is proposed.     

Electron microscopic immunocytological profiles in chronic fatigue syndrome

Fluoxetine is a 5-hydroxytryptamine (5-HT, serotonin)-selective reuptake inhibitor (SSRI) and is one of the main drugs used for the treatment of depression. Because it takes 2 to 3 weeks of treatment before clinical efficacy is manifest, the acute actions of fluoxetine cannot account for the clinical actions of the drug. The chronic effects of fluoxetine have not been completely delineated. The experiments detailed here investigate the chronic effects of fluoxetine on 5-HT and gamma-aminobutyric acid (GABA) receptor-mediated actions using intracellular recording techniques in hippocampal brain slices. Rats were treated with fluoxetine for 3 weeks via osmotic minipumps implanted s.c. Fluoxetine and norfluoxetine plasma levels were determined. The hippocampal pyramidal cell characteristics and the 5-HT1A and GABA(B) receptor-mediated hyperpolarization were measured in the CA1 and the CA3 subfields. The 5-HT4 receptor-mediated decrease in the slow afterhyperpolarization amplitude was also recorded in area CA1. The time constant, magnitude of the change in resistance during 300-ms hyperpolarizing current pulses and half-decay time of the sAHP were altered by chronic fluoxetine treatment in area CA1 pyramidal cells. No changes were seen in any of the active or passive membrane properties of the CA3 hippocampal pyramidal cells. Fluoxetine treatment increased the potency of 5-HT for the 5-HT1A receptor-mediated hyperpolarization in area CA1, but not area CA3, and decreased the potency of baclofen for the GABA(B) receptor-mediated hyperpolarization in area CA1, but not area CA3. The characteristics of the concentration-response curve for the 5-HT-mediated decrease in sAHP amplitude in area CA1 were not altered by fluoxetine treatment. Chronic fluoxetine selectively and differentially altered the cell characteristics and the 5-HT1A and GABA(B) receptor-mediated responses in area CA1 of the hippocampus, which forms the final common output of the hippocampus.     

Electron microscopic characterization of filiform papillae in the normal human tongue

The fine structure of filiform papillae on the normal human tongue was examined level by level, from the basal layer to the surface, in specimens taken from the dorsal side of the lingual body. Human lingual epithelia showed three distinct regions: epithelia on the anterior and on the posterior sides of filiform papillae and an interpapillary epithelium. While the basal and the squamous cell layers were similar throughout these three regions, differences were noted in the granular and the horny layers. The interpapillary epithelium actually lacked both the granular and the horny layers. The epithelium on the anterior side of filiform papillae was characterized by alternating layers of granular cells and of cornified cells. Granular cells possessed three types of keratohyaline-like granules within their cytoplasm: uniformly electron dense, relatively less electron dense, and a heterogeneous type. While the number of the keratohyaline-like granules was remarkably diminished in the epithelium on the posterior side of filiform papillae, a considerable amount of tonofibrils was present in the cytoplasm. In the uppermost portion of the anterior side of filiform papillae, coherence between adjacent epithelial cells depended mainly on the interlocking of cytoplasmic villi and poorly developed desmosomes on villi. On the other hand, epithelial cells on the posterior side of filiform papillae appeared to be more tightly adhesive compared with those on the anterior side. This was due to focal thickening of the plasma membrane and to desmosomes at the interface between the granular and cornified cells, and to the formation of a marginal band and increased intercellular cement presumably derived from lamellar bodies in the horny layer. These findings demonstrate distinct differences between the anterior and the posterior sides of filiform papillae in the human tongue with respect to keratinization patterns, structures associated with cell-to-cell adhesion and the strength of cellular cohesion in the uppermost portion, and the turnover of cornified cells. These differences may contribute to the formation of the unique external configurations of filiform papillae.