Kenzi Osanai

The cortical contraction related to the ooplasmic segregation inCiona intestinalis eggs

SummaryThe egg cytoplasm of ascidian,Ciona intestinalis, segregates towards both the animal and vegetal poles within a few minutes of fertilization or parthenogetic activation with ionophore A23187. A constriction appears first on the egg surface near the animal pole and then moves to the vegetal pole. Carmine granules and spermatozoa attached to the egg surface move towards the vegetal pole with the movement of the constriction. Microvilli, which are distributed uniformly in unfertilized egg, disappear on the animal side of the constriction and became more dense on the vegetal side of the constriction. Transmission electron microscopy revealed that sub-cortical cytoplasm, containing numerous mitochondria and sub-cortical granules, moves towards the vegetal pole with the movement of the constriction and then concentrates into a cytoplasmic cap at the vegetal pole. An electron-dense layer appears in the cortex of the cap. The ooplasmic segregation and the cortical contraction were inhibited by cytochalasin B and induced by ionophore A23187. These observations suggest that ooplasmic segregation is caused by the cortical contraction which is characterised by a surface constriction and by the formation of an electron-dense layer.

Distribution of actin filaments in fertilized egg of the ascidian Ciona intestinalis.

Microfilaments in the contracting cortex during the bipolar ooplasmic segregation of Ciona intestinalis eggs were examined by two methods, staining with fluorescent phalloidin and decoration with myosin subfragment 1 (S1). Fluorescent (Fl-)phalloidin revealed prominent fluorescence in the contracting cortex between the surface constriction and the vegetal pole of fertilized eggs. The animal pole did not stain. After extraction in Triton X-100, the cortex appeared as a thin layer that easily separated from cytoplasmic mass, especially at the contracting stage after fertilization. This layer also stained strongly with Fl-phalloidin. S1-decoration confirmed that actin filaments were abundant in the thin layer of Triton-extracted cortex. The actin filaments are considered to compose a contractile network covering the vegetal side of the constriction.

Cell Movements during Gastrulation of Starfish Larvae

Archenteron formation was monitored by measurement of cellular volume, injection of tracer enzyme, and vital staining. The cellular volume of the whole embryo did not change significantly from the start of gastrulation to the beginning of the mesenchyme-migration stage; the archenteron increased from about 10-20% during these stages. Tracer injection revealed that the boundary between the progenies of the veg1 and veg2 blastomeres of 32-cell-stage embryos was in the outer layer at the early gastrula stage, and at the rear end of the stomach at the bipinnaria stage. These results demonstrate a migration of cells from the outer layer to the archenteron wall during starfish gastrulation. Vital staining marks around the blastopore showed that the presumptive esophagus, stomach, and intestine area were added to the archenteron at the start of gastrulation, during the early to late gastrula stage, and thereafter, respectively. Tracer injection also indicated that the presumptive zone of the cardiac sphincter was twisted about 180{deg} clockwise around the axis of the archenteron after the late gastrula stage, dragging the cells in the presumptive zone of the esophagus and stomach.

Morphological identification of sperm receptors above egg microvilli in the polychaete, Neanthes japonica

A fine-structural study of sperm-egg interactions in the polychaete Neanthes japonica was carried out. Unfertilized eggs are surrounded by a chorion 0.6-0.7 micrometers thick. Oocyte microvilli are inserted into the inner layer of the chorion. The outer layers of the chorion are opened just above the tips of the microvilli, where a membrane vesicle (microvillus tip vesicle, about 0.2 micrometers in diameter) plugs the chorions opening. During fertilization, the acrosomal process of the sperm fuses with an egg microvillus within 1 min of insemination. All the microvillus tip vesicles disappear from the chorion surface within 5 min of insemination. When eggs, which are prefixed with glutaraldehyde, are inseminated, numerous sperm undergoing the acrosome reactions attach to the eggs. In the majority of these sperm, the tip of acrosomal process which is coated with the acrosomal content, adhere to a microvillus tip vesicle. These findings suggest that the microvillus tip vesicle serves as a sperm receptor, which induces the acrosome reactions and adhere to the sperm acrosomal process. The adhesion of the acrosomal process to the microvillus tip vesicle seems to be a prerequisite event for its fusion with the microvillus.

Cortical contraction and ooplasmic movement in centrifuged or artificially constricted eggs ofCiona intestinalis

SummaryTo discover the force causing bipolar ooplasmic segregation just after fertilization in ascidian eggs (Ciona intestinalis), cortical contraction and cytoplasmic movement were examined by centrifugation and by artificial constricting techniques. In the centrifuged eggs, the surface constriction appeared independently of cytoplasmic stratification. The yolk layer and the sub-centripetal layer moved toward the vegetal pole in the peripheral region. In the eggs which were artificially constricted by partially broken chorion and then fertilized, the inner cytoplasm always flowed from the vegetal sphere into the animal sphere during bipolar segregation. The direction of this cytoplasmic movement was independent of sphere size. This shows that the force causing cytoplasmic movement is supplied by the contraction of the vegetal-side cortex. It is suggested that the contracting cortex pushes the inner cytoplasm toward the animal pole and drags the peripheral cytoplasm toward the vegetal pole.

Structural and functional polarity of starfish blastomeres

The cortex of the blastomeres of Asterina pectinifera are structurally polarized so that some kinds of granules in the cortex, which can be stained vitally with Nile blue (Nile blue-positive granules, NBGs), and microvilli were distributed mainly in the apical region. The blastomeres always faced the adjoining blastomeres and blastocoel with the NBG-free, smooth region during embryogenesis. To confirm whether such blastomeres are functionally polarized, we rotated one of the blastomeres in the 2-cell-stage embryo so that it faced the other with the NBG-containing region. As a result, all embryos developed into twin or partitioned blastulae. This shows that the blastomeres are functionally polarized and have to orient the basal cortex toward the inner side of the embryo in order to be integrated into a blastula together with the others. The cortical polarity was formed and maintained even in blastomeres of dissociated embryos. In such blastomeres the cleavage furrows were formed along the axis of polarity. When the blastomeres began to adhere closely to each other at the 256-cell stage, only the NBG-free (basal) region acquired adhesiveness. These facts make it possible to infer why the correct apicobasal orientation of blastomeres is necessary for embryonic integration, without considering intercellular communication during the cleavage stage.

Chromosomal behavior in starfish (Asterina pectinifera) zygotes under the effect of aphidicolin, an inhibitor of DNA polymerase

When calf thymus histones were labeled fluorescently and microinjected into oocytes of the starfish, Asterina pectinifera, the labeled histones visualized chromosomes during maturation division and cleavage. In doing so, we confirmed the previously reported phenomenon that chromosomes became incompetent at the first cleavage in the aphidicolin-treated egg, although cleavage itself took place. Moreover, we found that chromosomes were aligned at the equator of the metaphase spindle of the first cleavage and that they did not separate into two groups at all, but made a lump in the middle of the spindle. Chromosomes finally entered one blastomere, although they did not participate in the following karyokinesis. DNA and microtubules were examined by cytochemistry and immunofluorescence in order to investigate the relation between chromosome movement and the microtubular cytoskeleton. The mitotic apparatus developed and grew in the aphidicolin-treated cells in the same manner as those in normal cells without normal chromatin condensation or chromosome movement during the first cleavage. However, the mitotic apparatus consisted of two asters without the spindle formed at subsequent cleavages. Electron microscopic study revealed that chromosomes did not condense normally and kinetochores were not detected during the first cleavage. These results indicate that the dynamic changes in microtubular structures during mitosis have poor relation with the chromosome behavior such as prophase chromosome condensation and anaphase chromosome movement.

Sperm attachment and acrosome reaction on the egg surface of the polychaete, Tylorrhynchus heterochaetus

Sperm binding to the egg envelope (chorion) was examined in fixed eggs and isolated chorions of the polychaete, Tylorrhynchus heterochaetus. Sperm binding included two successive steps: attachment (acrosomal outer surface-chorion binding) before the acrosome reaction and adhesion (acrosomal process-chorion binding) after the acrosome reaction. The attachment between sperm head-tip and the outermost layer of the chorion was observed in Ca-free seawater, in which the acrosome reaction did not occur. The surface of the chorion was stained with phosphotungstic acid (PTA). Sperm did not attach to pronase-treated eggs, in which the PTA-positive layer disappeared. When isolated chorions were soaked in distilled water for several hours, they lost the capacity for sperm attachment, and the PTA-positive layer thinned. The acrosome reaction was induced by material that was dissolved from the chorions into distilled water. This suggests that both the receptor for sperm attachment and the inducer of the acrosome reaction are involved in the PTA-positive layer.

Role of jelly matrix of egg masses in fertilization of the polychaete Lumbrineris latreilli

Summary A mature female of the polychaete Lumbrineris latreilli spawns an egg mass in which many unfertilized eggs are embedded in jelly matrix. Unfertilized eggs which were mechanically separated from the jelly matrix just after spawning lost the capacity for sperm binding, and therefore became unfertilizable. Fertilizability was restored by the addition of jelly fragments to the dejellied eggs. These results show that the jelly matrix is essential for successful fertilization, especially for establishing sperm-egg binding. Electron microscopy revealed that sperm-egg binding occurs between the head-tip of the acrosome-reacted spermatozoa and the outer surface of the chorion. The sperm acrosome reaction was not induced by the jelly matrix alone. Some interactions between the jelly matrix and spermatozoa may be prerequisite for induction of the acrosome reaction, which takes place on the outer margin of the chorion.

Cytochalasin B does not block sperm penetration into denuded starfish oocytes

During fertilisation in starfish oocytes, the fertilisation cone develops temporarily beneath the penetrating sperm. The role of the fertilisation cone in sperm incorporation in the starfish Asterias amurensis was examined using cytochalasin B (CB). CB (2 microM) allowed sperm acrosomal process-egg plasma membrane fusion and egg activation, but inhibited the development of the fertilisation cone containing actin microfilaments. When sperm were added to intact oocytes (with the jelly coat and vitelline coat) in seawater containing CB, the sperm head did not penetrate the fertilisation membrane. Although the acrosomal process fused with egg plasma membrane, the sperm head remained outside the fertilisation membrane. On the other hand, denuded oocytes without the jelly coat and vitelline coat allowed sperm penetration even in the presence of 2 microM CB. Electron microscopy revealed that sperm organelles, including the acrosomal process, nucleus, mitochondrion and tail, were incorporated into the slightly electron-dense cytoplasm, which was similar to the cytoplasm of the fertilisation cone. These results show that the development of the fertilisation cone/actin filament complex is not essential for incorporation of the sperm, since incorporation can occur in denuded oocytes. However, the cone is required for fertilisation of intact oocytes, suggesting that this actin-filament-containing structure is necessary for getting the sperm through the outer egg coats.