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.     

Ionic mechanism of the fertilization potential of the marine worm, Urechis caupo (Echiura)

Microelectrode and tracer flux studies of the Urechis egg during fertilization have shown: (a) insemination causes a fertilization potential; the membrane potential rises from an initial level of -33 +/- 6 mV to a peak at +51 +/- 6 mV (n = 16), falls to a plateau of about +30 mV, then returns to the original resting potential 9 +/- 1 min (n - 10) later; (b) the fertilization potential results from an increase in Na+ permeability, which is amplified during the first 15 s by a Ca++ action potential; (c) the maximum amplitude of the fertilization potential, excluding the first 15 s, changes by 51 mV for a 10-fold change in external [Na+]; (d) in the 10 min period after insemination, both Na+ and Ca++ influxes increase relative to unfertilized egg values by factors of 17 +/- 7 (n = 6) and 34 +/- 14 (n = 4), respectively; the absolute magnitude of the Na+ influx is 16 +/- 6 times larger than that of Ca++; (e) in the absence of sperm these same electrical and ionic events are elicited by trypsin; thus, the ion channels responsible must preexist in the unfertilized egg membrane; (f) increased Na+ influx under conditions of experimentally induced polyspermy indicates that during normal monospermic fertilization, only a fraction of available Na+ channels are opened; we conclude that these channels are sperm-gated; (g) Ca++ influx at fertilization is primarily via the membrane potential-gated channel, because kinetics are appropriate, and influx depends on potential in solutions of varying [Na+], but is independent of number of sperm incorporations in normal sea water.     

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.     

Fine structural investigation of the insemination response in Urechis caupo.

Electron microscopy of Urechis eggs revealed no changes in the egg cortex or investing layers until 4 min after insemination at 172C. From 4 min to about 30 min after insemination the surface coat gradually elevates, widening the perivitelline space. During this period, microvilli separate from the tightly woven layer of the surface coat, fibrogranular aggregates resembling surface coat material appear in the perivitelline space, and some cortical granules are extruded from the egg cortex into cytoplasmic processes. There is no statistically significant decrease in the number of cortical granules remaining in the egg surface during the first 95 min after insemination; many cortical granules persist in postgastrulae. Most of the cortical granules remain in fertilized eggs after removal of the surface coat with glucose-EGTA. We found no morphological correlates of the polyspermy block which is established within 1 min of insemination (Paul, 1975).     

Swimming behavior of the spoon worm Urechis unicinctus (Annelida,Echiura)

Large numbers of swimming and stranding Urechis unicinctus were observed at night during low tide in Sasuhama, Miyagi Prefecture, northeastern Japan, during the periods from January to February in 2012 and 2013. Worms did not drift passively but swam actively, therefore hinting at a certain purpose for such behavior. As trochophore larvae of U. unicinctus were observed to occur simultaneously in the plankton, we infer the possibility that this is an event of reproductive swarming. Anatomical observations of both swimming and stranding U. unicinctus showed that none of the specimens had gametes, which may suggest that these were completely spent after spawning. Urechis unicinctus seemed to begin swimming after dusk and the observed swimming behavior occurred during the evening ebb tide throughout the night low tide during winter time. Stranding U. unicinctus have long been known in Japan and have been attributed to sea storms. The present study shows for the first time the possibility that U. unicinctus swims in order to reproduce at night and that this swimming behavior is closely linked to the stranding of U. unicinctus individuals.     

The Sperm Proteome of the Echiuran Urechis unicinctus (Annelida,Echiura)

Sperm proteins presumably play critical roles in reproduction, but in many non‐model animals their identities are unknown. A total of 147 sperm proteins from the echiuran worm Urechis unicinctus, the first sperm proteome in the phylum Annelida, are reported. The echiuran sperm proteome can be classified into diverse functional groups: energy metabolism (31%), protein synthesis and degradation (18%), spermatogenesis and sperm motility (12%), signal pathway (11%), ion channel and transport proteins (6%), cytoskeleton (4%), immunity and stress responses (3%), and fertilization (1%). These results will facilitate studies of mechanisms of fertilization in echiurans, as well as comparative studies of reproduction and evolution across lophotrochozoans. Data are available via ProteomeXchange with identifier PXD009176.     

The polyspermy block in eggs of Urechis caupo: Evidence for a “rapid” block

Experiments have established the presence and time course of a polyspermy block in eggs of the echiuroid worm Urechis caupo. At various times after an initial insemination the eggs were reinseminated with concentrated sperm suspensions and the polyspermy determined. The block is rapid; partial protection against polyspermy is evident within a few seconds after insemination of the eggs. Although there are other rapid responses approximately correlated in time with the beginning of the block and possibly related to it, I also consider the possibility that a relatively ‘late’ event might account for the apparent ‘rapid’ block in these eggs.     

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.     

Screening of genes related to sulfide metabolism in Urechis unicinctus (Echiura,Urechidae) using suppression subtractive hybridization and cDNA microarray analysis

Exogenous sulfide can generally induce metabolic injuries in most organisms and even cause death. However, organisms inhabiting intertidal zones, hydrothermal vents, and cold seeps, can tolerate, metabolize, and utilize sulfide. In this study, both suppression subtractive hybridization and cDNA microarray analysis were employed to screen sulfide metabolism-related genes from the body wall in echiuran worm Urechis unicinctus, a marine sediment species. A total of 3456 monoclones were isolated and 82 were identified as differentially expressed genes in worms exposed to 50 μM sulfide for 24 h, compared to controls. The identified genes encoded proteins with multiple processes, including metabolism, cellular process, biological regulation, response to stimulus, multicellular organismal process, localization, development, and cellular component organization. Eight genes, serase, vacuolar protein, src tyrosine kinase, sulfide oxidase-like oxidoreductase, aprataxin, SN-RNP, aminopeptidase, and predicted protein, were selected to verify expression in the worm using qRT-PCR. The agreement of gene expression evaluation was 62.5% between the results of microarray analysis and qRT-PCR. These new data will provide clues for further probing of the molecular mechanism of sulfide metabolism.     

Cytochemical studies on the protamine-type protein transition in sperm nuclei after fertilization and the early embryonic histones of Urechis caupo.

Cytochemical staining characteristics of nuclear histones during postfertilization maturation division and various early embryonic stages in Urechis have been studied. The transition of protamine-type protein to adult histones in the sperm nucleus is accomplished by 15 min after entrance into the egg cytoplasm. Newly synthesized egg proteins migrate into enlarging male and female pronuclei after this transition, followed by pronuclear DNA synthesis and fusion. The shift from protamine-type protein to adult histones, which occurs in the absence of RNA synthesis during the postfertilization maturation division of the egg, may be one of the processes involved in the normal structural reorganization of chromosomes. Such a reorganization is likely to be a prerequisite for chromosome replication and mitosis. No qualitative differences are detected in the stainability of histones of unfertilized eggs and embryos at the cleavage and later stages of development.     

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.     

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.     

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.