Periodic Acid-thiosemicarbazide-silver Proteinate Research Articles

Heteromorphism of sperm axonemes in a parasitic flatworm, progenetic Diplocotyle olrikii Krabbe, 1874 (Cestoda, Spathebothriidea).

The spermatozoon ultrastructure of the progenetic cestode Diplocotyle olrikii (Spathebothriidea) has been examined using transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) for glycogen. The spermatozoon is a filiform cell, tapered at both extremities. Its moderately electron-dense cytoplasm possesses two parallel axonemes of unequal lengths. New for the Cestoda is a finding of three types of the mature spermatozoa with respect to different axonemal structure. The first type has both axonemes with standard 9 + '1' trepaxonematan pattern. The second type is represented by a spermatozoon having one axoneme with 9 + '1' structure and the second one with 9 + 0 pattern. The third type includes the two axonemes with 9 + 0 pattern. Microtubule doublets of the 9 + 0 axonemes contain either inner dynein arms or no dynein arms. In addition to the two axonemes, all three types of the mature sperm cells contain parallel nucleus, parallel cortical microtubules, four electron-dense plaques/attachment zones, and glycogen. The anterior extremity of the gamete exhibits a centriole surrounded by a semiarc of up to five electron-dense tubular structures. The distal end of the first type spermatozoa exhibits two morphological variants, represented either by (i) nucleus or (ii) remnants of the disorganized axoneme. Distal extremity of the spermatozoa of the second and third types contains doublets and singlets of disorganized axoneme. The ultrastructural characters of the spermatozoon of progenetic D. olrikii support the basal position of the Spathebothriidea within the Eucestoda.

Nippotaenia mogurndae Yamaguti et Myiata, 1940 (Cestoda, Nippotaeniidea): first data on spermiogenesis and sperm ultrastructure.

Spermiogenesis and the spermatozoon ultrastructure of the cestode Nippotaenia mogurndae Yamaguti et Myiata, 1940 (Nippotaeniidea), a parasite of Perccottus glenii Dubowski, 1877 (Perciformes: Odontobutidae), have been investigated by means of transmission electron microscopy, cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) for glycogen, and electron tomography. The process of spermatozoon formation is characterised by the presence of (1) two centrioles without typical striated rootlets, (2) a single intercentriolar body, (3) a flagellar rotation (free flagellum plus flagellar bud), and (4) a complete proximodistal fusion. The mature spermatozoon of N. mogurndae contains a single helicoidal crested body, one axoneme of the 9 + "1" trepaxonematan structure, parallel cortical microtubules arranged in a ring in the anterior region of the cell, and a spiraled nucleus encircling the axoneme. Intracellular components are situated in a moderately electron-dense cytoplasm, containing glycogen in the principal regions (II, III, IV) of the spermatozoon. Application of electron tomography has revealed a helicoidal nature of the central electron-dense core in the central cylinder of the axoneme in parasitic cestodes for the first time. The patterns of spermiogenesis and spermatozoon ultrastructure resemble most closely those in mesocestoidids and may reflect the relationships between Nippotaeniidea and Mesocestoididae.

Cytocomposition of the vitellarium in Khawia sinensis Hsü, 1935 (Cestoda, Caryophyllidea, Lytocestidae): another caryophyllidean species with lamellar bodies and lipids

The vitellarium of the invasive caryophyllidean tapeworm Khawia sinensis Hsü, 1935 from carp Cyprinus carpio L. was examined by means of transmission electron microscopy and cytochemical staining for glycogen with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP). A vitellarium consists of numerous follicles of irregular size that are interconnected by a net of vitelline ducts. Vitelline follicles are composed of vitelline cells at various stages of development that are interconnected by interstitial tissue. Vitelline follicles are surrounded by a cytoplasmic sheath associated with an intercellular matrix. Extensive development of the granular endoplasmic reticulum and Golgi complexes are both involved in the production of shell globules/shell globule clusters and characterise cytodifferentiation of vitellocytes. Nuclear and nucleolar transformation lead to the formation and storage of intranuclear glycogen, a feature specific for the Caryophyllidea. Newly observed within the mature vitellocytes of Khawia sp. is the presence of lamellar bodies and a few lipid droplets. These cytoplasmic inclusions first occur in the mature cells within the follicles and persist in the vitelline cells within vitelloducts and intrauterine eggs. Two types of lamellar bodies are detected: regular lamellar-structured body and irregular lamellar-structured body. None of the lamellar bodies are membrane bound. Results of the present study indicate that the formation of lamellar bodies may be closely related to the endoplasmic reticulum or shell globule clusters. Some of the shell globule clusters are transformed into lamellar body clusters. Ultrastructural features of vitellocytes in K. sinensis are compared with those of other monopleuroid, polypleuroid, and strobilated cestodes.

Reinvestigation of vitellogenesis in Caryophyllaeus laticeps (Pallas, 1781) (Cestoda, Caryophyllidea, Caryophyllaeidae), monozoic tapeworm of Abramis brama (Pisces, Teleostei)

Abstract Reinvestigation of vitellogenesis in the caryophyllidean cestode Caryophyllaeus laticeps (Pallas, 1781) has been performed using light microscope (LM) and transmission electron microscopy (TEM), and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) for glycogen. Vitellogenesis is generally similar to that reported in the past, however, some new observations were made. The present study reveals the first evidence of: (i) interstitial tissue in the vitelline follicles, (ii) lipid droplets in maturing and mature vitellocytes from vitelline follicles, and (iii) lamellar bodies in vitellocytes from the vitelloduct in C. laticeps. Projections of interstitial tissue surround each vitellocyte and the follicle periphery. The perinuclear cytoplasm of the interstitial tissue contains granular endoplasmic reticulum and vesicles of various size and density. Cytoplasmic osmiophobic lipid droplets and lamellar bodies, previously believed to be absent in most caryophyllid cestodes, are readily apparent in vitellocytes of C. laticeps. The origin and presumed function of these inclusions are discussed. On the other hand, the formation and storage of massive amounts of glycogen in the nucleus and large amounts in the cytoplasm of mature vitelline cells are similar to the condition found in other caryophyllids. Results are compared and contrasted with previous studies on vitellogenesis in other monopleuroid cestodes (Amphilinidea and Gyrocotylidea) as well as polypleuroid cestodes (Spathebothriidea) and the remaining strobilated Eucestoda.

Vitellogenesis in Archigetes sieboldi Leuckart, 1878 (Cestoda, Caryophyllidea, Caryophyllaeidae), an intestinal parasite of carp (Cyprinus carpio L.).

Vitellogenesis in the caryophyllidean tapeworm Archigetes sieboldi Leuckart, 1878, from carp Cyprinus carpio L. in Slovakia, has been examined using transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) for glycogen. Vitelline follicles extend in two lateral bands in the medullary parenchyma along both sides of the monozoic body. They are surrounded by an external basal lamina and contain vitellocytes and an interstitial tissue. The general pattern of vitellogenesis is essentially like that of other caryophyllideans. It involves four stages: immature, early maturing, advanced maturing cells and mature vitellocytes. During vitellogenesis, a continuous increase in cell volume is accompanied by an extensive development of cell components engaged in shell globule formation, e.g. granular endoplasmic reticulum and Golgi. Shell globule clusters are membrane-bound. Nuclear and nucleolar transformation are associated with formation and storage of large amounts of intranuclear glycogen, a very specific feature of the Caryophyllidea. For the first time, (a) additional vitelline material in Archigetes is represented by lamellar bodies and (b) lipid droplets are described in the mature vitellocytes from vitelline follicles and vitelloduct of the Caryophyllidea. Our results indicate that there may be a double origin of lamellar bodies: either from the endoplasmic reticulum or through transformation of shell globule/shell globule clusters. Lamellar body clusters and some single lamellar bodies appear to have a membrane. Other ultrastructural features of vitellogenesis and/or vitellocyte in A. sieboldi from its vertebrate (fish) and invertebrate (oligochaete) hosts are briefly compared and contrasted with those in other caryophyllideans and/or Neodermata.

Ultrastructural and cytochemical study on vitellogenesis in the diphyllidean cestode Echinobothrium euterpes (Echinobothriidae) and its phylogenetical implications

AbstractThe first description of vitellogenesis in the Diphyllidea is presented in this paper. Though the type of vitellogenesis and mature vitellocyte in Echinobothrium euterpes appear to be unique among the Eucestoda, however, they somewhat resemble that observed in the two orders of the lower cestodes, Tetraphyllidea and Proteocephalidea. Vitellocyte maturation is characterized by: (1) an increase in cell volume; (2) extensive development of short, parallel, frequently concentric cisternae of GER that produce dense proteinaceous granules; (3) development of Golgi complexes engaged in packaging this material; (4) progressive formation of saturated lipid droplets; their continuous enlargement and fusion; (5) formation of small accumulations of glycogen particles scattered between and among lipid droplets in the cytoplasm of maturing vitellocytes; (6) concentration of dense proteinaceous granules in the peripheral layer of cytoplasm, around the cell plasma membrane; and (7) vacuolization of cytoplasm of mature vitellocytes accompanied by a rapid increase in its volume. A new, unreported type of dense proteinaceous granules, situated around the limiting plasma membranes of mature vitellocytes, is described. Vitellogenesis evidently differs from that with typical shell-globules and shell-globule clusters previously reported in other taxa of lower cestodes. Cytochemical staining with periodic acidthiosemicarbazide-silver proteinate for glycogen indicates a strongly positive reaction for glycogen particles between and around large unsaturated lipid droplets of the maturing and mature vitellocytes. Some hypotheses concerning the interrelationship between this pattern of vitellogenesis, possible mode of egg formation, embryonic development and diphyllidean life cycle, and their phylogenetic implications are drawn and discussed.

Ultrastructure and cytochemistry of vitellogenesis in Wenyonia virilis Woodland, 1923 (Cestoda, Caryophyllidea)

AbstractVitellogenesis in Wenyonia virilis was examined by transmission electron microscopy (TEM), including the cytochemical detection of glycogen at the ultrastructural level with the periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) technique. Mature vitelline follicles have cells in various stages of development, progressing from immature cells of gonial type near the periphery of the follicle to maturing and mature vitellocytes towards the centre. Maturation is characterized by: (1) increase in cell volume; (2) increase in nuclear surface area restoring the N/C (nucleo-cytoplasmic) ratio; (3) nucleolar transformation; (4) extensive development of parallel cisternae of GER, the shell-protein producing units; (5) development of Golgi complexes, engaged in shell-granule/shell-globule formation and packaging; (6) synthesis and storage of glycogen in the cytoplasm; (7) simultaneous, independent formation and storage of intranuclear glycogen; (8) continuous fusion of small shell-granules into larger shell-globules and fusion of these into large shell-globule clusters with a progressive increase in the number and size of the latter; and (9) disintegration of GER in the medial layer of vitellocyte cytoplasm, degenerative changes and accumulation of glycogen and shell-globule clusters within the cytoplasm. The functional significance of numerous shell-globule clusters and the relatively small amount of nuclear and cytoplasmic glycogen is analysed. Unlike vitellogenesis of other caryophyllids, the nuclear glycogen of mature vitellocytes in W. virilis is randomly dispersed in the nucleoplasm and never forms a high central accumulation, the so-called “nuclear vacuole”. The nutritive function of vitellocytes appears greatly reduced in W. virilis, a fact perhaps related to the intrauterine development of the early embryos. The ultrastructure of vitellogenesis in W. virilis is compared with that in other lower cestodes, both monozoic and polyzoic. Conclusions concerning interrelationships of the vitellogenesis pattern of the ultrastructural cytochemistry of mature vitellocytes of W. virilis to intrauterine embryonation, absence of uterine glands and an extensive uterus characteristic for this species, are drawn and discussed.

Evidence for an intermediate type of spermatozoon: ultrastructural studies of spermiogenesis in the cuttlefishRossia macrosoma (Cephalopoda, Decabrachia)

Testes and spermatophores of the decapod cephalopodRossia macrosoma have been investigated by electron microscopy. Cytochemical analysis, by use of the periodic acid thiosemicarbazide-silver proteinate (PA-TSC-SP) technique, allows the precise localization of glycogen within the spermatozoon. Nuclear morphogenesis occurs in three stages (termed A, B and C), resulting in the transformation of the nucleus from an ovoid shape (stage A) into an elongated form (stages B and C). Prior to elongation of the spermatid nucleus, the spheroidal proacrosome, derived from the Golgi complex, becomes located at the apex of the nucleus. Later, the proacrosome elongates and its internal contents differentiate into an apical vesicle, electron-dense rod and electron-lucent subacrosomal fossa. A longitudinal microtubular manchette is closely associated with the elongating proacrosome. At this stage, the spermatid nucleus also elongates and its associated microtubular manchette is continuous with that surrounding the proacrosome. Large quantities of glycogen granules,β-particles, about 130–145 A in diameter, occur in the midpiece in relation to both the specialized external plasma membrane of the mitochondrial sleeve and within or around the axonome in the tail piece. We compare the features of spermiogenesis and the spermatozoon ofR. macrosoma with those documented in the literature for species from a number of phyla with the so-called primitive type of spermatozoon and which practise external fertilization, and the modified type in species practising internal fertilization. We suggest that the spermatozoon and spermiogenesis ofRossia demonstrates characteristics intermediate between these two groups and that the spermatozoon should be classified as an intermediate type.

Topographical localization of muscle glycogen: an ultrahistochemical study in the human vastus lateralis

The fine structural pattern of glycogen storage in resting and sprint-exercised human vastus lateralis muscle fibres of different types was analysed using ultrahistochemical methods. Three male subjects (31-36 years) performed 60 consecutive, supramaximal bouts of bicycle exercise, each starting every 1 min and having a duration of 8 s (including approximately 3 s of acceleration). The load was estimated to correspond to 200% of VO2-max. Five other subjects (22-27 years) constituted controls. Ultrathin sections stained with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) clearly revealed a compartmental distribution of glycogen. Glycogen is stored at five topographically, and probably also functionally, different locations. They are the subsarcolemmal, intermyofibrillar, para-Z-disc, N2-line, and H-zone spaces. During the exercise, glycogen from the N2-line and para-Z-disc locations is preferentially utilized. Serial sections stained with uranyl acetate and lead citrate demonstrated that glycogen stores of the type 2 fibres were more depleted than those of type 1 fibres. The implications of the differential intracellular glycogen storage are discussed.

An association of carbohydrates with particles of arabis mosaic virus retained within Xiphinema diversicaudatum

SUMMARYSections through the odontophore of Xiphinema diversicaudatum showed two types of staining for carbohydrates using the periodic acid ‐ thiosemicarbazide ‐silver proteinate (PA‐TSC‐SP) reaction. The first consisted of thin, localised, intensely‐stained patches on the lining of the food canal of all the specimens examined. The second type, found only in nematodes exposed to AMV‐infected plants, revealed cloud‐like areas of carbohydrate ‐ containing material associated with the stained patches on the lining of the food canal. By staining alternate sections with uranyl acetate/lead citrate, these carbohydrate clouds were shown to contain virus particles. Although the cloud material could have originated from either the plant or the nematode, sections through a pellet of partially purified virus particles prepared from plants did not stain for carbohydrate. The possible role of carbohydrate in virus retention and transmission is discussed.

Implementation of periodic acid-thiosemicarbazide-silver proteinate staining for ultrastructural assessment of muscle glycogen utilization during exercise.

Distribution of glycogen particles in semithin and ultrathin sections of biopsy samples from human muscles subjected to either short- or long-term running were investigated using PAS and Periodic Acid-ThioSemiCarbazide-Silver Proteinate (PA-TSC-SP) staining methods. Glycogen particles were predominantly found immediately under the sarcolemma or aligned along the myofibrillar I-band. After long-term exhaustive exercise type-1 fibers with a few or no glycogen particles in the core of the fibers were frequently observed. The subsarcolemmal glycogen stores of these "depleted" type-1 fibers were about three times as large as after exhaustive short-time exercise. Another indication of utilization of subsarcolemmal glycogen stores during anaerobic exercise was that many particles displayed a pale, rudimentary shape. This observation suggests fragmental metabolization of glycogen. Thus, depending on type of exercise and type of fiber differential and sequential glycogen utilization patterns can be observed.

A NEW ELECTRON MICROSCOPIC HISTO-CYTOCHEMICAL STAINING METHOD -DEMONSTRATION OF GLYCOGEN PARTICLES-

For electron microscopic demonstration of glycogen particles in tissue sections, a new histochemical staining method is introduced as a substitute for stains such as periodic acid thiosemicarbazide silver proteinate. It is based on the fact that glycogen particles can be demonstrated with alkaline bismuth (A·Bi) solution alone and without periodic acid pretreatment.A. The staining mechanism involves a direct reaction where vic-glycol groups in glycogen compounds are oxidized into carboxyl groups and bismuth is reduced. The latter combines with the carboxyl groups to effect increased electron opacity of glycogen particles under electron microscopic observations.B. The glycogen particles can be observed even if periodic acid treatment is added on condition that the treatment be stopped within 10-15min. But glycogen particles disappear when the treatment lasts more than 30min. The treatment is not necessary even in theory because the A·Bi stainable vic-glycol groups are gradually decreased in proportion to the duration of the treatment.C. The technique is adapted to the observation under high magnification of the mode of α-D-glucopyranose arrangement in glycogen compounds because the bismuth deposit has a high electron opacity due to the high atomic number of bismuth and is positioned only in the vic-glycol group of the glycogen compound.D. The staining technique is remarkably simple as staining needs to be carried out only once, in contrast with previous methods.This A·Bi staining technique permits excellent high contrast observation of glycogen particles in the cytoplasm of liver cells and myocardial muscle cells which could be seen to consist of fine granular substructures.

An ultrahistochemical study on glycogen in cow purkinje fibres

Ultrahistochemical staining with periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) indicated that cow Purkinje fibres are rich in glycogen in the form of round to oval particles, about 20 nm in diameter. Alignment or grouping of the glycogen particles was evident. Three types of 20 nm particles were distinguished by their different densities in sections stained with UAc and PbCi. The densest variety was interpreted to be ribosomes, and less dense particles in association with cytoplasmic filaments and very faintly stained particles between the filaments were considered to represent glycogen. The special character of glycogen in Purkinje fibres may be partly due to interactions between glycogen particles or between glycogen particles and filaments. Polysaccharide deposits were occasionally seen intercellularly, between Purkinje fibres. These deposits may be artefacts due to the preparative procedure, or to biological degeneration.

Ultrastructural localization of glycogen in erythrocytes and developing erythrocytic cells in normal human bone marrow.

The periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) reaction was employed for the ultrastructural cytochemical localization of saliva-labile glycogen in the erythrocytic cells in normal human blood and bone marrow. Particulate glycogen was demonstrated in the cytoplasm of all developmental forms of erythrocytic cells from the proerythroblast through the reticulocyte; a few particles of glycogen also were present in mature erythrocytes even in the peripheral blood. Statistical evaluation of the number of glycogen particles in mid-plane cell sections at each morphological stage of development indicated a significant and stepwise decrease during cellular maturation. This change in glycogen content may reflect both cellular utilization and mitosis during the maturational sequence.