Polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta. II. Large bleb formation caused by high concentrations of exogenous calcium ions

Fertilized eggs of the mollusk Ilyanassa obsoleta (Nassarius obsoletus) form large blebs resembling polar lobes within 12 min of exposure to solutions of isotonic CaCl₂, whereas control eggs in sea water remain spherical. Under identical conditions, fertilized eggs of the sea urchin, Strongylocentrotus purpuratus, an organism which normally does not form polar lobes, do not form blebs upon exposure to solutions of isotonic CaCl₂. The calcium-induced blebbing in Ilyanassa still occurs if other cations such as Na⁺, Mg²⁺, or Mn²⁺ are present in addition to Ca²⁺, but not if comparable concentrations of K⁺ are present. Cytochalasin B prevents the calcium-induced blebbing, whereas colchicine does not. Cytokinesis in both Ilyanassa and Strongylocentrotus and normal polar lobe formation in Ilyanassa appear to require exogenous K⁺ but not exogenous Ca²⁺. Preliminary electron microscopy of Ilyanassa eggs exposed to isotonic solutions of CaCl₂ has shown microfilaments in the cortical cytoplasm in the region of the bleb constriction but no microfilaments in spherical control eggs in sea water. These data suggest that high concentrations of exogenous Ca²⁺ trigger the polymerization and contraction of a ring of microfilaments in the cortical cytoplasm of the Ilyanassa egg which results in the formation of a lobelike bleb of cytoplasm. The observation that K⁺ antagonizes this Ca²⁺-induced blebbing has led to the formulation of a theory which postulates that the ratio of intracellular Ca²⁺ to intracellular K⁺ is critical in the control of polar lobe formation and cytokinesis.     

SEM observations on polar body elimination and changes of the oocyte surface during egg maturation in the newt Triturus alpestris (Amphibia,Urodela)

Summary We have studied the surface of the animal half of ovulated newt eggs recovered from different portions of the oviduct. The germinative area, about 40 m diameter, is localized in the region of the whitish polar spot, about 450 m diameter. The structural changes in the germinative area are connected with the formation and extrusion of the first and second polar bodies. Of the two types of oviductal eggs observed, those covered with microvilli (type 1) were found only in the ostial portions of the oviduct, whilst those covered with microfolds (type 11) were found more distally. The structural difference between these two types may be related to the known reduction in surface area of the cell membrane during oocyte maturation. Offprint requests to: W. Kilarski     

Cell subpopulations in the late morula and early blastocyst of the mouse

Late morulae and early blastocysts consist of two main cell subpopulations which occupy different positions within the embryos. The cells of the outer layer have a polar surface phenotype. The outward-facing surface of this cell type has a discrete dense pole of short microvilli, whilst the inward-facing surface has a relatively sparse distribution of longer, thick microvilli. The inner cells lack short, dense microvilli but exhibit thick microvilli of variable density. After short-term isolation in medium low in Ca²⁺, the individual polar and apolar cells remain distinguishable. The expanded blastocyst also has two major cell subpopulations, but within each of these, heterogeneity is observed. The mural trophectodermal cells have a larger, more regular outward-facing area of sparse, short microvilli than do polar trophectodermal cells. The ICM consists of some cells that show extensive blebbing in medium low in Ca²⁺ and others that do not.     

Polar effects of concanavalin A on the cortical cytoskeleton of a molluscan egg (Nassarius reticulatus,Gastropoda)

Summary The organization of the surface of fertilizedNassarius reticulatus eggs was probed by investigating the effects of treatment with concanavalin A (Con A). This lectin causes abnormal polar lobe formation as well as inhibition of cleavage. At low concentrations of Con A (0.3–1.0 μg/ml) the polar lobe constriction becomes considerably elongated, whereas at higher concentrations (2.5–50 μ/ml) the position of the constriction undergoes an extreme shift towards the animal pole. In the latter case, the surface of the animal part of the egg forms large protrusions and folds. Con A also causes resorption of microvilli and disappearance of the extracellular layer covering these villi; this process starts at the vegetal pole and propagates towards the animal pole. These changes in surface architecture are associated with profound changes in the organization of filamentous (F-) actin as assessed by confocal laser scanning microscopy of NBD-phallacidin-labelled eggs. Divalent succinyl-Con A has the same effects on polar lobe formation and surface architecture as tetravalent Con A, but only at very high concentrations (100–200 μg/ml), indicating that Con A exerts its effects by cross-linkage of its binding sites. Experiments with cytoskeleton inhibitors (cytochalasin D, colchicine, and nocodazole) reveal that in Con A-treated eggs — as in untreated eggs — microfilaments, but not microtubules, are involved in the formation of the polar lobe constriction. The calcium ion channel blocker D600 affects neither normal nor Con A-induced abnormal polar lobe formation, which suggests that influx of external calcium is not required. In contrast, treatment with TMB-8, an antagonist of internal calcium release, prevents the formation of a polar lobe in both normal and Con A-treated eggs. Finally, eggs from which the polar lobe has been removed prior to Con A treatment show none of the effects described, whereas isolated polar lobes react similarly to intact eggs. These results suggest that binding of Con A to sites present at the vegetal pole of the egg is responsible for the observed effects of the lectin.     

Surface ultrastructure of paddlefish eggs before and after fertilization

The surface ultrastructure of eggs of the paddlefish Polyodon spathula was investigated by scanning electron microscopy. Mature eggs of paddlefish possess four to 12 micropyles in the animal polar region. There are sperm entry sites in the egg surface under the micropyles which consist of tufts of microvilli. Five to nine sperm entry sites were observed on mature eggs. Probably, the number of sperm entry sites corresponds to the number of micropyles. In a few eggs, 1 min after fertilization the ball-like enlarged top of a cytoplasmic process (probably a full-grown fertilization cone) had reached the external aperture or the canal of several micropyles. In other micropyles of the same egg, a few smaller cytoplasmic processes or flocculent material were found in the micropylar canal. With one exception, no sperm tails were found there. The formation of the full-grown cytoplasmic process is possibly initiated before the cortical reaction has started in an area of the animal hemisphere. Three, 10 and 20 min after fertilization, the uneven surface of the cortical cytoplasm in the animal polar region rose gently where microvilli were much less than the in other area and together with a secondary polar body at the latter stage. Taken together, paddlefish eggs may have sperm entry sites corresponding to the number of micropyles and respond to the stimulus of fertilization by forming a few cytoplasmic processes–fertilization cones (larger and smaller). Sperm penetration into the egg may be achieved at an earlier stage of fertilization (sperm-egg contact), as inferred from the fact that a secondary polar body was formed at the 20-min stage irrespective of the exceptional finding of the sperm tail.     

Freeze-fracture characteristics of the ocellar retina of the honeybee,Apis mellifica carnica pollm. (Hymenoptera : Apidae)

Freeze-fracture electron microscopy has been utilized to investigate membrane specializations in the dark-adapted retina of the ocellus of worker honeybee, Apis mellifica (Hymenoptera : Apidae).The distal segment of the photoreceptor cell is unilaterally composed of short microvilli, termed rhabdomeres. All microvilli are oriented in the same direction. Transversally fractured rhabdomerss show the typical hexagonal arrangement of the microvillar profiles. The plasma membrane limiting each microvillus, is rich in particles (4000/μm² on the protoplasmic (P) face). Two kinds of P-face particles are seen: one type is about 4nm in diameter and the other has diameters of 8 – 10nm. Because of their size and density, these particles may mainly correspond to photopigments. Plaques of exoplasmic (E) face particles present in the glial cells sheathing the photoreceptor cells, are thought to represent gap junctions. Special differentiations of the smooth endoplasmic reticulum (ER) are found in the perirhabdomeric cytoplasm closely associated with microvilli. The membranes of the ER are wound up to form large concentric bodies. These bodies may play an important role in formation of new microvilli. Furthermore, conspisuous bodies with stomata are found in the perirhabdomeric cytoplasm.     

Quantitative cellular analysis of growth and reproduction in freshwater planarians (Turbellaria; Tricladida). I. A cellular description of the intact organism

Summary

Changes in egg shape and surface morphology during maturation may be related to the localization of cytoplasmic determinants in the embryos of organisms with spiral cleavage. The eggs of the polyclad turbellarian Hoploplana inquilino undergo pronounced shape changes during the meiotic divisions which have been examined with the scanning electron microscope. Unfertilized eggs have a smooth surface that becomes covered with microvilli and microblebs within 10 min of fertilization. First polar body extrusion is accompanied by the asymmetric appearance of large blebs (Blebbing Cycle I) primarily in the animal hemisphere with one quadrant characteristically smoother than the others and bearing fewer blebs. Blebbing Cycle II, which is less pronounced than the first but is still characterized by a relatively un blebbed quadrant of the zygote, coincides with second polar body formation. These asymmetric shape changes in the animal hemisphere during meiosis may possibly correlate with a primitive form of morphogenetic segregation and beginning quadrant specialization in polyclads, the most primitive spiralians with mosaic development.     

Effect of Microvilli on Lateral Diffusion Measurements Made by the Fluorescence Photobleaching Recovery Technique

To consider the effect of surface microvilli on measurements of lateral diffusion by fluorescence photobleaching recovery, we have measured the diffusion of the lipid probe 3,3′-dihexadecyl indocarbocyanine iodide on the villated main body and unvillated budding polar body of unfertilized mouse eggs. On the main body we found D = (6.41 ± 0.62) × 10^-9 cm²/s with (77.0 ± 2.1)% recovery, and on the budding polar body we found D = (7.05 ± 0.75) × 10^-9 cm²/s with (84.7 ± 1.3)% recovery. We thus find only slight differences in diffusion in the two regions.     

Fine Structure of the Surface of Small Mucous Cells in the Epidermis of the Hagfish Myxine glutinosa (Cyclostomata)

Scanning and transmission electron microscopic investigations of the epidermal surface cells, the small mucous cells, of Myxine glutinosa reveal specific structural differentiations in the form of vertical apical channels with microvilli and supporting tonofilament skeleton. The functional significance of these surface differentiations with regard to the living environment of Myxine is discussed and a comparison with the surface characteristics of other aquatic animals is drawn.     

Food Uptake by the Insect-Parasitic Nematode,Sphaerularia bombi (Tylenchida)

The insect-parasitic female of Sphaerularia bombi everts its uterus and associated reproductive structures into the body cavity of its bumblebee host. This uterine sac then takes over the normal functions of the parasite and leads an independent existence. An examination of this sac shows that the surface of the uterine cells are differentiated into a network of saccular indentations separating off fine cytoplasmic extensions. The folding of adjacent cytoplasmic extensions around portions of the host''s hemolymph results in the formation of pinocytotic vacuoles. Intracellular vacuoles are also formed at the base of the saccular indentations. It appears that the first stage of intracellular digestion in S. bombi initially occurs by pinocytosis in the outer surface of the uterine cells. The inner and outer surfaces of the ovary and oviduct are modified into lobelike projections to increase the absorptive surface area, and electron-dense droplets originating in this tissue were also observed in the developing eggs. The hypothesis is presented that soluble nutrients are passed into the ovary-oviduct tissue where, after being reconstituted into droplets, they enter the developing eggs.     

Scanning and transmission electron microscopic studies of jejunal microvilli of the rat, hamster and dog

Scanning and transmission electron microscopy were used to investigate the surface area and number per unit area of microvilli from jejunal villus epithelial cells in the rat, hamster and dog. The calculated mean microvillus surface area was 0.419 μ², 0.573 μ², 0.751 μ² for the rat, hamster and dog respectively. The largest number of microvilli per square micron freeze dried villus surface was measured in the rat with a mean value of 65. Hamster and dog freeze dried specimens had lower mean values of 54 and 34 microvilli respectively. The total microvilli surface area in square micron per square micron villus surface was more closely related for the three species with values of 27.23 for the rat, 30.94 for the hamster and 25.53 for the dog. These data indicate an inverse relationship between the mean microvillus surface area and population density in the species studied. However, the total microvilli surface area per unit villus surface is relatively similar for the three species. The observed number of microvilli per unit villus surface was shown to vary depending upon the dehydration technique employed for preparation of scanning electron microscopic specimens. This variation probably reflects shrinkage artifact and should be considered in soft tissue studies involving the scanning electron microscope.     

Ultrastructural changes induced by juvenile hormone analogue in oöcyte membranes of apterous4Drosophila melanogaster

Egg chambers from apterous⁴ (ap⁴), a female sterile mutant of Drosophila melanogaster, show none of the microvilli or pinocytotic vesicles which are a prominent feature of the membrane of the wild-type vitellogenic oöcyte. The studies reported here show that a juvenile hormone analogue (ZR515) stimulates formation of microvilli and pinocytotic vesicles in oöcytes of ap⁴ flies. Within 12 hr after topical application of ZR515 to homozygous ap⁴ females the oöcyte membranes exhibit extensive microvilli and pinocytotic activity. The follicle-cell surface adjoining the oöcyte also shows some changes. In vitro studies in which ap⁴ ovaries were incubated in Schneider's Drosophila tissue-culture medium in the presence of ZR515 with or without female haemolymph, or in the absence of ZR515, showed that the analogue acts alone directly on the ovary to cause formation of microvilli and pinocytotic vesicles on the oöcyte membrane.     

Microiontophoretic injection of calcium ions or of cyclic AMP causes rapid shape changes in fertilized eggs of Ilyanassa obsoleta.

Microiontophoretic injection of calcium ions or of adenosine 3′:5′-cyclic monophosphoric acid (3′:5′-cAMP) causes fertilized eggs of Ilyanassa obsoleta to form a large lobe-like protuberance near the micropipet tip within 15–30 sec. A protuberance can be induced to form anywhere on the egg surface, i.e., animal hemisphere as well as vegetal hemisphere. Injections of comparable amounts of Na⁺, K⁺, Mg²⁺, Hepes buffer, seawater, guanosine 2′:3′-cyclic monophosphoric acid (2′:3′-cGMP), or guanosine 3′:5′-cyclic monophosphoric acid (3′:5′-cGMP) have no effect on cell shape. Injection of 2′:3′-cAMP causes slight changes in cell shape. Injection of Ca²⁺ generates a shape change in spherical eggs, as well as during all phases of normal polar lobe formation, but not when polar lobes are being resorbed. Injection of Ca²⁺ elicits a shape change only when injection currents exceed 120 nA and only when Ca²⁺ also is present in the exogenous bath solution. Cell shape changes causes by injection of 3′:5′-cAMP also are dependent upon a minimum current (approximately 300 nA) and upon the presence of exogenous Ca²⁺. These shape changes may depend upon exogenous Ca²⁺ either because the injections trigger a change in membrane permeability, or because exposure of eggs to Ca²⁺-free seawater lowers intracellular [Ca²⁺] to such an extent that threshold levels of Ca²⁺ are not attained during injection.     

Scanning and transmission electron microscopic studies of the surface of Vallisia indica (Monogenea,Polyopisthocotylea)

Ultrastructural studies were carried out on the surface of Vallisia indica, found on the gills of Scomberoides commersonianus in the Indian ocean. Tegumental serrations present on the hump and on the dorsal and ventro-lateral surfaces of the hindbody lie in the same plane as the secondary gill lamellae and may provide anchorage against displacement forces. Elsewhere the tegument is highly folded forming a reticulum of lamellae and microvilli. The surface syncytium of the tegument in the region of the serrations possesses fibrous electron-dense bars, and a continuous fibrous electron-dense terminal web is present beneath the apical plasma membrane in all regions. The haptor consists of two asymmetrical rows of pincer-type clamps and a terminal lappet with a pair of hooks. Each clamp may be withdrawn into a cushion-like pad. Uniciliated sensory structures are found around the mouth and on the body surface, whereas non-ciliated papillae-like structures are present on the haptor and hindbody. A pair of cone-like sensilla is present on the lappet.     

On some species of microfossils from the Torgin formation of the Neoproterozoic of the Berezov Depression (Southern Siberian Platform)

A new genus, Soktokuta (Rhodophyta), and new species S. sporifera, Talakania diversa, Torgia munientis, and T. ellipsoidea are described. The diagnosis of the genus Torgia Grigorieva et Kolosov is emended. The genus Torgia is classified within the Chlorophyta. The Late Riphean (Neoproterozoic) beds of the South Urals and Siberia contain ellipsoidal unicellular fronds (Chlorophyta) with polar structures (dense and frequently spiny terminations).     

Temperature‐mediated trade‐offs and changes in life‐history integration in two slipper limpets (Gastropoda: Calyptraeidae) with planktotrophic development

Intraspecific variation in egg size and hatching size, and the genetic and environmental trade‐offs that contribute to variation, are the basis of the evolution of life histories. The present study examined both univariate and multivariate temperature‐mediated plasticity of life‐history traits, as well as temperature‐mediated trade‐offs in egg size and clutch size, in two planktotrophic species of marine slipper limpets, Crepidula. Previous work with two species of Crepidula with large eggs and lecithotrophic development has shown a significant effect of temperature on egg size and hatching size. To further examine the effect of temperature on egg size in Crepidula, the effects of temperature on egg size and hatching size, as well as the possible trade‐offs with other the life‐history features, were examined for two planktotrophic species: Crepidula incurva and Crepidula cf. marginalis. Field‐collected juveniles were raised at 23 or 28 °C and egg size, hatching size, capsules/brood, eggs/capsule, time to hatch, interbrood interval, and final body weight were recorded. Consistent with results for the lecithotrophic Crepidula, egg size and hatching size decreased with temperature in the planktotrophic species. The affects of maternal identity and individual brood account for more than half of the intraspecific variation in egg size and hatching size. Temperature also showed a significant effect on reproductive rate, with time to hatch and interbrood interval both decreasing with increasing temperature. However, temperature had contrasting effects on the number of offspring. Crepidula cf. marginalis has significantly more eggs/capsule and therefore more eggs per brood at 28 °C compared to 23 °C, although capsules/brood did not vary with temperature. Crepidula incurva, on the other hand, produced significantly more capsules/brood and more eggs per brood at the lower temperature, whereas the number of eggs/capsule did not vary with temperature. The phenotypic variance–covariance matrix of life‐history variables showed a greater response to temperature in C. incurva than in C. cf. marginalis, and temperature induced trade‐offs between offspring size and number differ between the species. These differences suggest that temperature changes as a result of seasonal upwelling along the coast of Panama will effect the reproduction and evolution of life histories of these two co‐occurring species differently. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

A new species of Myrcia (Myrteae, Myrtaceae) from the Atlantic Forest of southern Bahia, Brazil

Myrcia thomasii, a new species from the Atlantic Forest of southern Bahia, is described and illustrated. The new species is similar to M. micropetala, but differs in having the inflorescence and basal bracts puberulent, lanceolate bracts at the base of flowers, longer calyx lobes and petals, and a 3-locular ovary.     

Control of Polar Lobe Formation in Fertilized Eggs of Ilyanassa obsoleta Stimpson

Mechanisms of polar lobe formation in fertilized eggs of Ilyanassaobsoleta are discussed. Data are presented in the context ofa model involving a contractile ring of microfilaments in thecortical cytoplasm of the vegetal hemisphere. The polar lobeneck diameter decreases at two distinct rates during formationof second, third, and fourth polar lobes. During the second,more rapid phase of lobe constriction, eggs contain a band ofmicrofilaments arranged circumferentially in the cortical cytoplasmapposed to the plasma membrane at the base of the polar lobeconstriction. These microfilaments disappear and lobe constrictionsregress in cytochalasin B, but not in colchicine. Colchicineprevents eggs from beginning the second, more rapid phase oflobe constriction. Eggs require the presence of potassium ionsbut not sodium ions for normal polar lobe formation and cleavage.When eggs are placed in isotonic solutions of CaCl₂, within10–15 min they form cytoplasmic blebs which enlarge intolobes. This calcium-induced blebbing is inhibited by cytochalasinB but not by colchicine. Blebbing occurs in the calcium concentrationrange of 0.17 M-0.34 M in the presence of NaCl, MgCl₂, or MnCl₂.Potassium ions actively inhibit the calcium-induced blebbing,however.     

A Three-dimensional model reconstruction of pole assembly in Bacillus subtilis

In the rod-shaped bacterium Bacillus subtilis, new polar surfaces arise at division through the centripetal synthesis of a centrally located cross-wall. Subsequently, the cross-wall, analogous to a flat annulus, is converted into two inner layers of polar wall as the daughter cells separate. The junction of polar and cylindrical wall is marked by the presence of raised tears or wall bands formed by the splitting apart of the cross-wall at its base. New polar wall formed in this manner accounts for about 15% of the total surface area. The sequence of pole formation has been simulated by means of a generalized conic section based upon the mathematical rotation of a parabola about its longitudinal axis. Four basic measurements describe the stages of pole formation with reference to polar surface area: the equatorial diameter at the wall bands (D_max), the division furrow (D_min), the horizontal distance (h) from the centre of the cross-wall to D_max and the curvature of the nascent polar surfaces. These four parameters were found to yield a close fit to measurements of polar size and shape derived from electron micrographs of cell poles in sectioned organisms. Calculations of pole curvature suggest that both the initial separation of the cross-wall and separation of the daughter cells may occur very rapidly.