Regulation of histone synthesis during early Urechis caupo (Echiura) development

The histones present in mature oocytes and embryos of Urechis caupo and their pattern of synthesis during early development have been characterized. Acid-soluble proteins extracted from mature oocyte germinal vesicles and from embryonic nuclei were analyzed by two-dimensional polyacrylamide gel electrophoresis. Histones are accumulated in the mature oocytes in amounts sufficient to provide for the assembly of chromatin through the 32- to 64-cell stage of embryogenesis. Two H1 histones, which appear to be variants, were found. Germinal vesicles and cleavage-stage nuclei are enriched in H1M (maternal). During late cleavage a faster-migrating H1, H1E (embryonic), appears among the nuclear histones and, as embryogenesis continues, replaces H1M as the predominant H1. No new core histone variants are detected during early development. Examination of [³H]lysine-labeled histones from germinal vesicles and embryonic nuclei reveals stage-specific patterns of histone synthesis. H1M is the major H1 species synthesized in mature oocytes. After fertilization, a switch to the predominant synthesis of H1E occurs. Comparison of the [³H]lysine incorporated into H1E and core histones indicates that H1E synthesis is disproportionately high from midcleavage through the midblastula stage. By the gastrula stage, a balanced synthesis of H1E and each core histone is established. The results indicate that there is noncoordinate regulation of H1 and core histone synthesis during Urechis development.     

Parthenogenesis in Urechis caupo (Echiura) II. Role of intracellular pH in parthenogenesis induction

A peptide (P23) isolated from sperm acrosomal protein initiates development in eggs of the marine worm Urechis caupo . We have shown previously that eggs exposed to P23 for ≥3min complete meiosis but fail to cleave. However, a brief (1.5–2 min) exposure to P23 at pH 8, followed by either acidification of the seawater to pH 7 or dilution of P23 at pH 8 causes germinal vesicle breakdown (GVBD), but eggs fail to complete meiosis and many then later advance to mitosis. In the present study we investigated the hypothesis that partial activation leading to parthenogenesis occurs when there is a partial intracellular alkalinization. Measurements with the fluorescent pH indicator bis(carboxyethyl)-carboxyfluorescein (BCECF) showed that P23 induces a pH, increase similar to that occurring during fertilization and that parthenogenesis-inducing treatments interupt this rise in pH₁ In eggs exposed to P23 for >3 min the pH₁ increase was 0.31–0.49 units, slightly higher than in fertilized eggs. In partially activated eggs exposed to P23 for 1.5–2 min at pH 8, pH₁ began to rise but then returned to control values or remained only partially elevated (< 0.2 pH units average increase). Electrophysiological measurements revealed that removal of P23 during the first few minutes of exposure caused the activation potential to terminate and experiments with [¹⁴C]-P23 confirmed that dilution results in a rapid unbinding of P23 from eggs. If proton export is driven by membrane potential as well as the pH gradient, these results explain why dilution of P23 at pH 8 also interrupts the pH_i increase.     

Metameric organisation of the nervous system in developmental stages of Urechis caupo (Echiura) and its phylogenetic implications

The phylogenetic position of Echiura is still in continuous debate. The commonly accepted view regards Echiura as a distinct taxon, often classified as phylum, which forms the sister group of the Articulata. The alternative view considers Echiura to be a subtaxon of Annelida, which is supported by numerous shared characters. The correct systematic position of Echiura is inevitably linked to the presence or absence of true segmentation. The apparent lack of segmentation in Echiura is considered to be either primary, thereby supporting their exclusion from Annelida, or alternatively to be the result of reduction. The latter would clearly substantiate their classification as a subtaxon of Annelida. Immunohistochemical methods and confocal laser-scanning microscopy clearly demonstrate a metameric organisation of the nervous system in different larval stages of Urechis caupo, which corresponds to the segmental arrangement of ganglia in "typical" Annelida. This segmental pattern is reflected in the serially repetitive distribution of neurons containing the neurotransmitter serotonin (5-hydroxytryptamine) and also in the corresponding distribution of strictly paired peripheral nerves. Precisely two pairs of peripheral nerves are associated with each of the repetitive units. This metameric pattern also corresponds to the transient annulation of the trunk, which is found in late larval stages. Other characters of the nervous system including the paired origin of the ventral nerve cord, the anterior-posterior development gradient and the presence of a distinct suboesophageal ganglion are also found accordingly in typical Annelida. These results are interpreted as an indication that Echiura are derived from formerly segmented ancestors, and thus support their systematic inclusion within Annelida.     

Release of acid and changes in light-scattering properties following fertilization of Urechis caupo eggs.

The release of a fertilization acid, monitored by measuring the pH of egg suspensions, begins within 10 sec of insemination of Urechis caupo eggs. This is 4 min before the vitelline layer begins to elevate and is apparently unrelated to that process. The eggs of two molluscs, Mytilus californianus and Acmaea incessa, do not form a fertilization acid. The acid of Urechis eggs is not accompanied by release of “fertilization” carbohydrate, sulfate, or a nonvolatile weak acid into the seawater. The light-scattering properties of Urechis eggs change during the first 10 min after insemination. A decrease in light scattering begins by 10 sec and is complete by 1 min (Phase I). This is followed by a further decrease (3–6 min, Phase II) and an increase (6–10 min, Phase III). In striking contrast to an overtly similar situation in sea urchin eggs (fertilization acid and coincident light-scattering decrease), the release of acid and the initial light-scattering change are not the result of cortical granule discharge, and the acid, at least, is not related to the changes in shape or surface area which the eggs undergo. The processes underlying these rapid events are not yet known.     

Studies of oogenesis in Urechis caupo. I. Method for separating different size classes of oocytes

The immature oocytes of the echiuroid worm Urechis caupo develop while suspended as single cells in the coelomic fluid, free of any associated nurse or follicle cells. In any one female, size classes can be found ranging from 8 to 130 μm in diameter. A method is described for obtaining four to five relatively uniform size fractions by centrifuging the mixed oocyte population through a discontinuous Ficoll gradient and collecting the different size oocytes which accumulate at each step of the gradient. Phagocytosis of oocytes in vivo is also discussed.     

Cloned myogenic cells during differentiation: membrane biochemistry and fine structural observations.

Isolated membranes from clones of a permanent line of myogenic cells, L₆, were studied during three temporally consecutive stages of growth: (1) exponentially growing cells, (2) intermediate phase cells, and (3) fused cells. Membrane fractions have been isolated on a sucrose gradient and studied with respect to morphology, enzymatic activity, density, and α-bungarotoxin binding. Changes as a function of differentiation were seen both in the density of the fractions and in the specific activities of several proteins. Membranes characterized by specific enzyme markers generally sediment at higher densities when isolated from exponentially growing cells than when isolated from fused cells. The change in density is especially pronounced for the 5′ nucleotidase, TPNH-dependent cytochrome c reductase, and β-N-acetylglucosaminidase. The specific activity of adenylate cyclase increases in the intermediate phase cells and remains high in the fused cells; that of TPNH-dependent cytochrome c reductase decreases in the intermediate phase cells and remains low in the fused cells. The binding of α-bungarotoxin increases dramatically following fusion. On the other hand, there was little change, less than a factor of two, in the specific activities of a number of enzymes during growth or following fusion. In this group are hexose-6-phosphatase, acid phosphatase, Na⁺,K⁺-activated ATPase, and 5′ nucleotidase. Both our morphological and our biochemical studies suggest that in the exponentially growing cells, the ribosomes are associated largely with membranes, whereas in the fused cells, the ribosomes are largely free.     

Spontaneous feline sporotrichosis: A fine structural study

Fine structural details of the parasitic yeastlike phase of Sporothrix schenckii contained in biopsy tissue from a naturally-occurring case of disseminated feline sporotrichosis are described and illustrated by electron microscopy. Both free and phagocytosed fungal cells were observed. The fungal cells were contained within an extracellular, electron transparent vacuolar area which was bounded by a limiting membrane of probable host origin. The yeastlike cells were characterized by a conspicuous layer of osmiophilic microfilaments which occurred along the outermost surface of the cell wall. In many yeastlike cells, scattered, membranebound vacuoles containing electron opaque material were observed in the cytoplasm. Asteroid bodies were not observed.     

A cytological study of the ovary of Rhodnius prolixus. II. Oocyte differentiation

The masticatory apparatus of the vespertilionid bat Myotis lucifugus appears generalized. Principal modifications for more efficient trituration have involved accessory tooth cusps. Chewing strokes pass through orbits (up to 7/sec) involving translations along and rotation about three axes. Direction of chewing typically reverses by at least the fifth or sixth consecutive orbit. Reversal involves modification of the downstroke at varying positions along its course. Compared to certain other bats, which do not utilize oral phonation for echolocation, Myotis chews much more rapidly, with many more degrees of freedom in orbital configuration. The overall envelope of motion is remarkably similar in all these species. The jaw muscles of Myotis act asymetrically, and in more than one direction as the orbit progresses. They overlap in their periods of activity forming a continually-modified muscular sling. Unilateral force couples facilitate orthal rotation at the condyles and movements of them. Bilateral couples, pitting protrusors of one side against retrusors of the other, facilitate lateral translation. The pterygoids are instrumental in continuing motion across the top and bottom of the orbit. Countercontractions are particularly important in stabilizing and protecting the temporomandibular joints. The mandibular symphyseal joint appears to act passively, providing additional flexibility for the system. Higher nervous control beyond the simple jaw-opening reflex appears necessary to explain the firing order of the digastrics and the phase relationship of orbital reversal to overall muscular firing intensity. Control mechanisms, ancillary phenomena and comparative aspects are discussed.     

Initial stages of tunic morphogenesis in the ascidian Halocynthia: a fine structural study

Tunic morphogenesis in embryos of the ascidian Halocynthia roretzi was examined by scanning and transmission electron microscopy. For this purpose it was necessary to modify the classical embedding procedure. Soon after reaching the initial tail-bud stage, tunic deposition is initiated on the dorsal side of the embryo. As soon as the embryo is completely covered by the tunic, larval fins are formed. The test cells settle onto the embryo. At this stage only the outer cuticle and the outer tunic compartment have appeared. Tunic morphogenesis is accompanied by ultrastructural modifications of the epidermis characteristic of secreting cells. Cytochemical investigations reveal polysaccharide glycogen-like material in the lumen of epidermal lacunae and in the outer compartment of the tunic. Our observations strongly suggest that this material is stored in the lacunae and discharged into the outer compartment. The significance of fluffy osmiophilic material that appears at the early tail-bud stage and enlaces the whole embryo is discussed.     

Gonadotrophs following removal of the ovaries: a fine structural study of human pituitary glands.

The fine structure of gonadotrophs has been investigated in surgically removed pituitary glands of 12 women who because of disseminated breast cancer, underwent bilateral ovariectomy at various periods before hypophysectomy. Compared with the adenohypophyses of 3 non-ovariectomized female subjects with diabetes mellitus, electron microscopy revealed that two cell types were affected by gonadectomy. These cell types corresponded to those which were regarded as FSH gonadotrophs and LH gonadotrophs in previous studies. In addition in the adenohypophyses stimulated by removal of the ovaries, intermediary cell types began to appear suggesting a transformation of LH gonadotrophs to FSH gonadotrophs. The most conspicuous change following gonadectomy was the formation of castration cells. These cells arose from FSH gonadotrophs and exhibited ultrastructural features interpreted as representing the morphologic manifestations of sustained hypersecretion of gonadotrophins. It seemed that castration cells have a limited life span and in their advanced stages of development they show ultrastructural signs indicative of irreversible involution.     

Homology and Evolution of the Chaetae in Echiura (Annelida)

Echiura is traditionally regarded as a small phylum of unsegmented spiralian worms. Molecular analyses, however, provide unquestionable evidence that Echiura are derived annelids that lost segmentation. Like annelids, echiurans possess chaetae, a single ventral pair in all species and one or two additional caudal hemi-circles of chaetae in two subgroups, but their evolutionary origin and affiliation to annelid chaetae are unresolved. Since annelids possess segmental pairs of dorsal (notopodial) and ventral (neuropodial) chaetae that are arranged in a row, the ventral chaetae in Echiura either represent a single or a paired neuropodial group of chaetae, while the caudal circle may represent fused rows of chaetae. In annelids, chaetogenesis is generally restricted to the ventral part of the notopodial chaetal sac and to the dorsal part of the neuropodial chaetal sac. We used the exact position of the chaetal formation site in the echiuran species, Thalassema thalassemum (Pallas, 1766) and Echiurus echiurus (Pallas, 1767), to test different hypotheses of the evolution of echiurid chaetae. As in annelids, a single chaetoblast is responsible for chaetogenesis in both species. Each chaeta of the ventral pair arises from its own chaetal sac and possesses a lateral formation site, evidencing that the pair of ventral chaetae in Echiura is homologous to a pair of neuropodia that fused on the ventral side, while the notopodia were reduced. Both caudal hemi-circles of chaetae in Echiurus echiurus are composed of several individual chaetal sacs, each with its own formative site. This finding argues against a homology of these hemi-circles of chaetae and annelids’ rows of chaetae and leads to the hypothesis that the caudal chaetal rings evolved once within the Echiura by multiplication of ventral chaetae.     

Organ culture of adult amphibian heart: a fine structural analysis.

Pieces of hearts from adult newts were cultured up to 2 months. Within 7 days of culture, approximately 37% of the cardiac explants were attached to the substrate and more than 33% of the attached explants and approximately 15% of the unattached explants established pulsation rates ranging from 3 to 67 beats/min. The control and cultured explants were processed at weekly intervals for electron microscopy. The diameter of the control cardiac muscle cells ranged approximately 3-5 micron. The cell surface was provided with microvilli. The intercellular spaces ranged approximately 150-500 A. The intercalated discs lacked the step-like courses observed in the mammalian cardiac muscle. Sarcoplasmic reticulum was scanty. Desmosomal-dense materials were frequently continuous with the Z-bands of both control and cultured cardiac muscle cells. The transverse tubular system and gap junction were absent in newt ventricles. The functional implications of these characterisitics are discussed. At the end of 1 week of culture, the surfaces of the explants were covered by one or more layers of non-muscle cells, and the core of the explants consisted mostly of cardiac muscle cells. In a few cardiac muscle cells the myofibrillar organization was disrupted, resulting in the distribution of scattered patches of myofibrils and free myofilaments in the sarcoplasm. A small number of intact muscle cells contained a considerable number of dense granules in the sarcoplasm. At 15 days in culture, a large number of muscle cells showed structural features reminiscent of embryonic cardiac muscle cells. These cells possessed patches of myofibrils, scattered myofilaments and scanty sarcoplasmic reticulum along with other cellular organelles and inclusions. Several of these altered cardiac muscle cells contained mitotic figures. The cardiac explants maintained the initial beating rate until the end of 2 months of culture, except for the 11% of the explants which stopped beating. By 3-4 weeks in culture, most of the cardiac muscle cells possessed the altered cell morphology mentioned above. The explants after 60 days in culture became more flattened than the earlier explants. The intact cardiac muscle cells were rare, and the cores of the explants were mostly occupied by the altered cardiac muscle cells. It is evident from our studies that the cardiac muscle cells have undergone dedifferentiation in long-term culture, and that this dedifferentiation process has yet had no effect in the maintenance of contractility of the explants. Furthermore, these dedifferentiated cardiac muscle cells are capable of DNA synthesis and mitosis.     

Some fine structural observations on the rhizome ofDendroligotrichum (Bryophyta)

Summary The underground portion (rhizome) of the gametophytic axis ofD. dendroides bears anatomical resemblances to a triarch dicotyledonous root. The similarities include: 1. an epidermis producing epidermal appendages; 2. a cortex with endodermoid layer; and 3. a tri-radiate arrangement of the food and water-conducting tissue. Histochemical observations reveal that the entire radial and transverse walls of the endodermoid cells are encrusted with amorphous deposits, probably of polyphenolic nature. Casparian bands are not present as reported by earlier workers. The radial walls exhibit a fine structure of alternating electron-dense and electron-opaque lamellae. In plasmolyzed cells the plasmalemma does not adhere to the radial wall. Plasmodesmatal connections were observed in the radial and outer tangential walls of the endodermoid cells, but not in the inner tangential walls. These features of the endodermoid layer ofD. dendroides are discussed in relationship to the structure and function of the endodermis of vascular plants.     

Comparative study of enzymes in testes and ovaries from adult Dipetalogaster maximus (Uhler) and triatoma infestans (Klug) (Hemiptera: Reduviidae). correlation with fine structural organization.

Activities of hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PDH), fructose-6-phosphate kinase (F6PK), glutamate dehydrogenase (GlutDH), aspartate aminotransferase (AAT), malate dehydrogenase (MDH) and glycerol-3-phosphate dehydrogenase (GPDH) were determined in tissue extracts of testes and ovaries of adult Dipetalogaster maximus (Uhler) and Triatoma infestans (Klug) (Hemiptera: Reduviidae), insect vectors of Chagas disease. The fine structure organization of the same organs were studied by electron microscopy. Results allow the following inferences: in testes from both species, most of the glucose would be utilized through the glycolytic pathway. Amino acid catabolism for energy purposes appears to be unimportant. The number of mitochondria and the development of the rough endoplasmic reticulum in cells of the spermatogenic line indicate the occurrence of active oxidative metabolism and protein synthesis; in ovaries, levels of G6PDH indicate the existence of an active pentose pathway which would supply the NADPH required for fat and ecdysteroid synthesis. Amino acid catabolism appears to be relatively more important in ovary than in testis. Fat and glycogen are stored in follicular cells of D. maximus; oocytes of both species contain numerous fat droplets. Abundant mitocondria are present in follicular cells and oocytes. A well developed rough endoplasmic reticulum and free ribosomes are also conspicuous in these cells. The malate/aspartate H-transfer system seemed to be relatively more important than the glycerophosphate shuttle in ovaries as well in testes.     

High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study

A new technique employing continuous recirculating perfusion of the rat liver in situ, shaking of the liver in buffer in vitro, and filtration of the tissue through nylon mesh, results in the conversion of about 50% of the liver into intact, isolated parenchymal cells. The perfusion media consist of: (a) calcium-free Hanks'' solution containing 0.05% collagenase and 0.10% hyaluronidase, and (b) magnesium and calcium-free Hanks'' solution containing 2 mM ethylenediaminetetraacetate. Biochemical and morphologic studies indicate that the isolated cells are viable. They respire in a medium containing calcium ions, synthesize glucose from lactate, are impermeable to inulin, do not stain with trypan blue, and retain their structural integrity. Electron microscopy of biopsies taken during and after perfusion reveals that desmosomes are quickly cleaved. Hemidesmosome-containing areas of the cell membrane invaginate and appear to pinch off and migrate centrally. Tight and gap junctions, however, persist on the intact, isolated cells, retaining small segments of cytoplasm from formerly apposing parenchymal cells. Cells which do not retain tight and gap junctions display swelling of Golgi vacuoles and vacuoles in the peripheral cytoplasm. Cytoplasmic vacuolization in a small percentage of cells and potassium loss are the only indications of cell injury detected. By other parameters measured, the isolated cells are comparable to normal hepatic parenchymal cells in situ in appearance and function.