Gamete interactions and the fate of sperm organelles in fertilized echinoderm eggs

Investigations of gamete fusion, sperm entry and the fate of the sperm nucleus, plasma membrane, mitochondrion, and axonemal complex in fertilized echinoderm eggs are reviewed. The timing of gamete fusion with respect to the onset of electrical activity characteristic of the activated egg and the affects of fixation conditions on the stability of fusing membranes are discussed. Observations from investigations using cationized ferritin labeled gametes and immunogold cytochemistry to demonstrate the mixing of sperm plasma membrane components within the egg plasma membrane, in particular along the surface of the fertilization cone, are compared with results from studies in somatic cells. Transformations of the sperm nucleus into a male pronucleus, consisting of sperm nuclear envelope breakdown, chromatin dispersion, and formation of a pronuclear envelope, are correlated with recent biochemical observation of similar processes in other cellular systems. Fates of the sperm mitochondrion and axonemal complex are examined.     

Structure and function of the extracellular matrix of anuran eggs

The extracellular matrix (ECM) surrounding the anuran egg is composed of jelly coat layers, an envelope, and the perivitelline space, which separates the envelope from the egg plasma membrane. Both the jelly coat layers and egg envelopes are required for fertilization in anurans. This paper reviews the current understanding of the structure-function relations of the ECM, with emphasis on the egg envelope. The fibrous egg envelope exists in four related forms. The envelope forms differ in their ultrastructures, macromolecular compositions, and cellular functions. After the oocyte is released from the ovary, conversion of one envelope form to another is brought about by factors secreted by the oviduct prior to fertilization and by factors released from the egg in the sperm-triggered cortical reaction. An additional extracellular matrix structure, located in the perivitelline space, has recently been identified in Xenopus laevis, as well as a previously undescribed reorganization of envelope fibers occurring at fertilization. The molecular changes in the ECM glycoproteins (limited proteolysis, lectin-ligand binding, and conformational changes) and the oviductal and egg macromolecules responsible for the conversion of envelope forms are discussed. New experimental evidence that supports the lectin-ligand hypothesis for the formation of the fertilization layer is presented. It is proposed that the molecular changes in the ECM are responsible for the ultrastructural alterations of the ECM and for modifications of the fertilization and developmental functions of the anuran egg ECM.     

Transformation of the amphibian oocyte into the egg: Structural and biochemical events

Amphibian oocytes, arrested in prophase I, are stimulated to progress to metaphase II by progesterone. This process is referred to as meiotic maturation and transforms the oocyte, which cannot support the early events of embryogenesis, into the egg, which can. Meiotic maturation entails global reorganization of cell ultrastructure: In the cell cortex, the plasma membrane flattens and the cortical granules undergo redistribution. In the cell periphery, the annulate lamellae disassemble and the mitochondria become dispersed. In the cell interior, the germinal vesicle becomes disassembled and the meiotic spindles form. Marked changes in the cytoskeleton and mRNA distribution also occur throughout the cell. All of these events are temporally correlated with intracellular signalling events: Fluctuations in cAMP levels, changes in pH, phosphorylation and dephosphorylation, and ion flux changes. Evidence suggests that specific intracellular signals are responsible for specific reorganizations of ultrastructure and mRNA distribution.     

Rotary shadowing with platinum-carbon in biological electron microscopy: A review of methods and applications

Rotary shadowing when combined with such specimen preparation techniques as quick freezing and deep etching, critical point drying, and glycerol spraying is a highly versatile method of visualizing cell and macromolecular ultrastructure. This review outlines the procedures commonly used to prepare specimens for rotary shadowing and evaluates the relative merits of rotary shadowing when compared to unidirectional shadowing and negative staining.     

Angiogenesis in wound repair: angiogenic growth factors and the extracellular matrix

Angiogenesis is critical to wound repair. Newly formed blood vessels participate in provisional granulation tissue formation and provide nutrition and oxygen to growing tissues. In addition, inflammatory cells require the interaction with and transmigration through the endothelial basement membrane to enter the site of injury. Angiogenesis, in response to tissue injury, is a dynamic process that is highly regulated by signals from both serum and the surrounding extracellular matrix (ECM) environment. Vascular endothelial growth factor, angiopoietin, fibroblast growth factor, and transforming growth factor beta are among those most potent angiogenic cytokines in wound angiogenesis. The cooperative regulation of them is essential for wound repair. Migration of endothelial cells and development of new capillary vessels during wound repair is dependent on not only the cells and cytokines present but also the production and organization of ECM components both in granulation tissue and in endothelial basement membrane. The ECM regulates angiogenesis by providing scaffold support and signaling roles. They also serve as a reservoir and modulator for growth factors. Laminins are the major noncollagenous ECM of endothelial basement membrane. Two newly recognized laminins, 8 and 10, are the major laminins produced by human dermal microvascular endothelial cells. Laminin 10 is highly expressed in blood vessels around skin wounds. Laminin 8 promotes dermal endothelial cell attachment, migration, and tubule formation. Integrins with either beta 1 or alpha v subunits are the major cellular surface receptors for ECM molecules and mediate the interactions between cells and ECM during wound angiogenesis.     

Comparative ultrastructure of the fertilized egg envelope in Corydoras adolfoi and Corydoras sterbai,Callichthyidae, Teleostei

In teleost, the structural characteristics of fertilized egg and egg envelope are very important for classification of genus or species. The structures of fertilized egg and egg envelope from Corydoras adolfoi and Corydoras sterbai, Callichthyidae, Siluriformes in teleost were examined by scanning and transmission electron microscopes to confirm whether these morphological structures have specificities of species and family or not. The fertilized eggs of C. adolfoi and C. sterbai were non‐transparent, spherical, demersal, and strong adhesive. There were no structural differences between two species through the light microscope. The size of the fertilized eggs of C. adolfoi was 1.95 ± 0.03 mm (n = 20), and that of C. sterbai was 1.92 ± 0.03 mm (n = 20). The perivitelline space was almost not developed in both species. In both species, the adhesive protuberances structures were on the outer surface of egg envelope. And fibrous structures were specially located at attachment part of spawning bed. And the egg envelope consisted of two layers, an inner lamellae layer and an outer strong adhesive layer with high electron dense protuberances structures in cross section. Consequentially, the fertilized eggs, outer surface on the egg envelope and cross section of egg envelope have identical structure. So, these structural characteristics of fertilized eggs and egg envelope show genus Corydoras specificity.     

A new HPF specimen carrier adapter for the use of high‐pressure freezing with cryoscanning electron microscope: two applications: stearic acid organization in a hydroxypropyl methylcellulose matrix and mice myocardium

Cryogenic transmission electron microscopy of high‐pressure freezing (HPF) samples is a well‐established technique for the analysis of liquid containing specimens. This technique enables observation without removing water or other volatile components. The HPF technique is less used in scanning electron microscopy (SEM) due to the lack of a suitable HPF specimen carrier adapter. The traditional SEM cryotransfer system (PP3000T Quorum Laughton, East Sussex, UK; Alto Gatan, Pleasanton, CA, USA) usually uses nitrogen slush. Unfortunately, and unlike HPF, nitrogen slush produces water crystal artefacts. So, we propose a new HPF specimen carrier adapter for sample transfer from HPF system to cryogenic‐scanning electronic microscope (Cryo‐SEM). The new transfer system is validated using technical two applications, a stearic acid in hydroxypropyl methylcellulose solution and mice myocardium. Preservation of samples is suitable in both cases. Cryo‐SEM examination of HPF samples enables a good correlation between acid stearic liquid concentration and acid stearic occupation surface (only for homogeneous solution). For biological samples as myocardium, cytoplasmic structures of cardiomyocyte are easily recognized with adequate preservation of organelle contacts and inner cell organization. We expect this new HPF specimen carrier adapter would enable more SEM‐studies using HPF.     

Microscopical evaluation of extracellular matrix and its relation to the palatopharyngeal muscle in obstructive sleep apnea

Obstructive sleep apnea hypopnea syndrome (SAHS) is a complex disease of the upper respiratory airways. SAHS physiopathology is multifactorial in which airway compliance is a very important component. To evaluate the tissue changes in the palatopharyngeal muscle by morphometric, histochemical, immunohistochemical, and stereological quantification, with special attention to extracellular matrix associated with this muscle at the structural and ultrastructural levels. Thirty patients with SAHS were divided into groups of 10 according to disease severity: mild, moderate, and severe SAHS. In addition, the control group consisted of 10 patients. Fragments of palatopharyngeal muscle removed from patients with SAHS and tonsillectomies from patients in the control group were histopathologically submitted to light microscopy and transmission electron microscopy. Histopathological evaluations by light and transmission electron microscopes showed differences in analyzed groups, such as reduction of the muscle fiber diameter in patients with SAHS, taking disease severity into consideration. In contrast, stereological analysis showed a gradual increase of the collagen and elastic system fibers relative frequencies, proportionally to SAHS seriousness. MMP‐2 and MMP‐9 immunostaining also showed an increased reaction in the muscle fiber cytoplasm and endomisium during SAHS progression. The ultrastructural analysis showed that palatopharyngeal muscle fibers presented cytoplasmic residual corpuscles, a sign of early cell aging. In conclusion, the increase of tissue compliance in individuals with SAHS can be, in addition to other factors, consequence of diminished contractile activity of the muscle fibers, which exhibited clear signs of early senescence. Moreover, extracellular matrix components changes may contribute to muscle myopathy during SAHS progression. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Histochemical and structural characterization of egg extra‐cellular matrix in bufonid toads,Bufo bufo and Bufotes balearicus: Molecular diversity versus morphological uniformity

The extra‐cellular matrix of fertilized eggs in the bufonid toads Bufo bufo and Bufotes balearicus was studied to clear the relationships between structural and molecular diversity. Histochemical (PAS, AB pH 2.5 and pH 1.0, Beta‐elimination PAS) and lectin‐histochemical (Con A, WGA, Succinyl‐WGA, PNA, RCA‐1, DBA, SBA, AAA, UEA‐I, LTA) techniques were used and the observations were made under light and electron microscopy. Both species present a fertilization envelope (FE) and two jelly layers (J₁ and J₂). The fibers of J₂ are shared among the eggs of a clutch in a jelly ribbon. The FE of both species presents neutral glycoproteins, mostly N‐linked. In B. bufo there are also residuals of mannose and/or glucose and N‐acetylglucosamine. In the FE fibers run parallel to egg's surface or are in bundles or looser hanks with no clear orientation. The J₁ layer of both species presents sialosulfoglycoproteins, mostly O‐linked, with lactosaminylated, galactosaminylated, glycosaminylated, and fucosylated residuals. A lower amount of galactosaminylated residuals is observed in B. balearicus in respect to B. bufo, whereas the opposite is seen in the amount of fucosylated residuals. The J₂ layer is similar in composition to J₁ but in B. balearicus there are no glucosaminylated residuals. J layers present fibers and granules that reduce towards J₂. Several microorganisms, in particular blue algae, are observed in the J₂ layer of both species. In respect to other species, B. bufo and B. balearicus have a lower number of jelly layers, but a comparable number of glycan types. Microsc. Res. Tech. 77:910–917, 2014. © 2014 Wiley Periodicals, Inc.     

Regeneration of spines and pedicellariae in echinoderms: a review.

Morphogenesis of tissues during regeneration of echinoderm spines and pedicellariae is reviewed. Regeneration of the skeleton is rather well documented while that of associated soft tissues is poorly investigated. In particular, little information is available on the early regeneration stages which follow wound healing. From the available information, it is suggested that regeneration of broken spines proceeds through a morphallactic process of which the organizational information, as well as the involved cells, lies in the stump. In contrast, regeneration of removed spines and pedicellariae may depend on an epimorphic process whose organizational information could be located in the mutable connective tissue that joins the appendage to the main body wall.     

Application of an envelope technique in the detection of ball bearing defects in a laboratory experiment

To judge the diagnostic capabilities of an envelope technique, based on enveloping the resonance frequency of a transducer,

1 Bearcon Signature™, Carl Schenck AG, Germany

artificially damaged ball bearings (SKF 6207) were run under oil lubrication at different radial loads and speeds in a laboratory experiment. Damage to the outer race, inner race and ball of different magnitudes was introduced by spark erosion and the response of the measurement system was analysed. The results showed that vibration spectrum components of outer race defects showed up in the envelope spectra of undamaged bearings, and that the detectability of defects was primarily limited by the speed of the bearing; on occasion the automatic scaling facility of the analyser was a limiting factor. Inner race and ball defects showed the well-known effects of load modulation in the defect contact zone, which can become dominant in the envelope spectra at high loads and speeds. This phenomenon could be confirmed by computer simulation. The detection of ball damage was hampered by the fact that overrolling of the defect in purely radially loaded bearings takes place incidentally. This drawback could be mitigated by applying a trigger technique to the envelope signal.     

Determining the composition of small features in atom probe: bcc Cu-rich precipitates in an Fe-rich matrix

Aberrations in the ion trajectories near the specimen surface are an important factor in the spatial resolution of the atom probe technique. Near the boundary between two phases with dissimilar evaporation fields, ion trajectory overlaps may occur, leading to a biased measurement of composition in the vicinity of this interface. In the case of very small second-phase precipitates, the region affected by trajectory overlaps may extend to the centre of the precipitate prohibiting a direct measurement of composition. A method of quantifying the aberrant matrix contribution and thus estimating the underlying composition is presented. This method is applied to the Fe–Cu-alloy system, where the precipitation of low-nanometre size Cu-rich precipitates is of considerable technical importance in a number of materials applications. It is shown definitively that there is a non-zero underlying level of Fe within precipitates formed upon thermal ageing, which is augmented and masked by trajectory overlaps. The concentration of Fe in the precipitate phase is shown to be a function of ageing temperature. An estimate of the underlying Fe level is made, which is at lower levels than commonly reported by atom probe investigations.     

Fine structure and formation of the eggshell in scorpionfly Panorpa liui Hua (Mecoptera: Panorpidae)

The morphology and formation of the eggshell in the scorpionfly Panorpa liui Hua were examined with light microscopy and scanning and transmission electron microscopy. During oogenesis of the scorpionfly, the follicle cells multiply by mitotic divisions and diversify into four morphologically distinct subpopulations, three of which are engaged in the eggshell formation and mold various parts of chorion. The eggshell consists of three layers: An inner vitelline membrane, an outer chorion, and a precursor extrachorion. The chorion constitutes a very compact endochorion, a rough fibrillar exochorion, and a polygonal meshwork of elevated ridges. At proximal end of ovarioles the chorion of matured oocyte is covered with a loose membrane, which might be the remnant of follicle cells. The jelly substance, which acts as lubricant to protect the oviducts and ovulated eggs during ovulation, might add to the top of polygonal ridges as the outermost extrachorion after oviposition. The eggshell formation process in Panorpa is tentatively proposed. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc.     

Scanning and transmission electron microscopy study of the microstructural changes occurring in aluminium matrix composites reinforced with SiC particles during casting and welding: interface reactions

Processing of aluminium matrix composites (AMCs), especially those constituted by a reactive system such as Al–SiC, presents great difficulties which limit their potential applications. The interface reactivity between SiC and molten Al generates an aluminium carbide which degrades the composite properties. Scanning and transmission electron microscopes equipped with energy-dispersive X-ray spectroscopes are essential tools for determining the structure and chemistry of the Al–SiC interfaces in AMCs and changes occurring during casting and arc welding. In the present work, an aluminium–copper alloy (AA2014) reinforced with three different percentages of SiC particles was subjected to controlled remelting tests, at temperatures in the range 750–900 °C for 10 and 30 min. Arc welding tests using a tungsten intert gas with power inputs in the range 850–2000 W were also carried out. The results of these studies showed that during remelting there is preferential SiC particle consumption with formation of Al₄C₃ by interface reaction between the solid SiC particle and the molten aluminium matrix. The formation of Al₄C₃ by the same mechanism has also been detected in molten pools of arc welded composites. However, in this case there was formation of an almost continuous layer of Al₄C₃, which protects the particle against further consumption, and formation of aciculate aluminium carbide on the top weld. Both are formed by fusion and dissolution of the SiC in molten aluminium followed by reaction and precipitation of the Al₄C₃ during cooling.     

Freeze-substitution: the addition of water to polar solvents enhances the retention of structure and acts at temperatures around –60°C

High‐pressure freezing followed by freeze substitution and plastic embedding is becoming a more widely used method for TEM sample preparation. Here, we have investigated the influence of solvents, fixative concentrations and water content in the substitution medium on the sample quality of high‐pressure frozen, freeze‐substituted and plastic embedded mammalian cell culture monolayers. We found that the visibility of structural details was optimal with acetone and that extraction increased with both increasing and decreasing solvent polarity. Interestingly, the addition of water to polar solvents increased the sample quality, while being destructive when added to apolar solvents. The positive effect of water addition is saturable in acetone and ethanol at 5%(v/v), but even addition of up to 20% water has no negative effect on the sample structure. Therefore, a medium based on acetone containing fixatives and 5% water is most optimal for the substitution of mammalian cell cultures. In addition, our results suggest that the presence of water is critical for the retention of structure at temperatures around –60°C.     

New elements in the interreceptor matrix: a comparative study of Megavilli and Landolt's club

Megavilli and Landolt's clubs were reported in the interreceptor matrix of the retina of the goldfish and neonatal rat for the first time. Megavilli encroach onto the inner or outer segments of photoreceptors and were different from microvilli. Landolt's clubs were not present in the adult rat but only in the adult goldfish.     

An investigation of the effect of changes in engine operating conditions on ignition in an hcci engine

The dependence of the ignition timing in an HCCI engine on intake temperature and pressure, equivalence ratio, and fuel species is investigated with a zero-dimensional model combined with a detailed chemical kinetics. The accuracy of the model is evaluated by comparing measured and computed results in a propane-fueled HCCI engine. It is shown that the peak pressure values are reproduced within 10% and ignition timing within 5° CA. The heat loss through the walls is found to affect significantly on the ignition timing for different inlet conditions. It is also shown that for the propane-fueled engine, the tolerance in intake temperatures is 20–25K and the tolerance in intake pressure is about 1 bar for stable operation without misfire or too early ignition. Comparison of propane and heptane fuels indicates that the tendency to misfire when heptane is employed as the fuel is less than that when propane is employed with the same wall temperature conditions. However, the heptane-fueled engine may have a Lower compression ratio to avoid too early ignition and hence lower efficiency. For the selected set of engine parameters, stable operations might be achieved for the heptane-fueled engine with twice as much tolerance in intake temperatures as for the propane-fueled engine.     

A new approach for high-pressure freezing of primary culture cells: the fine structure and stimulation-associated transformation of cultured rabbit gastric parietal cells

A newly designated procedure for high‐pressure freezing of primary culture cells provided excellent ultrastructure of rabbit gastric parietal cells. The isolated parietal cells were cultivated on Matrigel‐coated aluminium plates for conventional subsequential cryoimmobilization by high‐pressure freezing. The ultrastructure of different organelles (Golgi apparatus, mitochondria, multivesicular bodies, etc.) was well preserved compared to conventional chemical fixation. In detail, actin filaments were clearly shown within the microvilli and the subapical cytoplasm. Another striking finding on the cytoskeleton system is the abundance of microtubules among the tubulovesicles. Interestingly, some microtubules appeared to be associating with tubulovesicles. A large number of electron‐dense coated pits and vesicles were observed around the apical membrane vacuoles in cimetidine‐treated resting parietal cells, consistent with an active membrane uptake in the resting state. Immunogold labelling of H⁺/K⁺‐ATPase was seen on the tubulovesicular membranes. When stimulated with histamine, the cultured parietal cells undergo morphological transformation, resulting in great expansion of apical membrane vacuoles. Immunogold labelling of H⁺/K⁺‐ATPase was present not only on the microvilli of expanded apical plasma membrane vacuoles but also in the electron‐dense coated pits. The present findings provide a clue to vesicular membrane trafficking in cultured gastric parietal cells, and assure the utility of the new procedure for high‐pressure freezing of primary culture cells.     

The fine structure and morphogenesis of the envelope and nucleocapsid of goat poxvirus and infectious bovine rhinotracheitis virus: A freeze-fracture study

We studied the morphology and morphogenesis of viral envelopes and nucleocapsids of goat poxvirus (GPV) and infectious bovine rhinotracheitis virus (IBRV) by means of the freeze-fracture technique. The GPV at an early development stage was fractured in its middle, and the envelope was shown to be a bilayer of particles. The mature GPV was fractured between two monolayers of the envelope. These facts suggest that there is little lipid and mainly protein particles in the envelope at the early-development stage, then the lipid inserts into the envelope during viral development. We found that there were still many intramembranous protein particles in protoplasmic fracture face (PF) and extracellular fracture face (EF) of the envelope of mature GPV in the cytoplasm, and fewer particles in the envelope of released GPV. In the envelope of mature IBRV, however, there were many more intramembrane protein particles in the PF face than that in the EF face. Spike-like structures could be seen at the outer edge of the IBRV envelope at times. Protein particles were regularly arranged in the plasmic membranes contacting IBRV. This phenomenon seems to be related to IBRV release. The naked cores, empty capsids, and nucleocapsids of IBRV were assembled in the nucleus of infected cells at the same time. The assembled nucleocapsids could be divided into five types according to their different fracturing positions, whose morphology was observed after deep etching. The morphology of the samples prepared with different methods was compared as well.