Miyako S. Hamaguchi

Analysis of the Role of Astral Rays in Pronuclear Migration in Sand Dollar Eggs by the Colcemid-UV Method

The formation and migration of the sperm aster, and the migration of male and female pronuclei during fertilization were investigated in the eggs of the sand dollar, Clypeaster japonicus using the Colcemid‐UV method. When an egg in Colcemid sea water was irradiated locally with UV light (about 365 nm wavelength) at a limited region containing sperm head, a sperm aster formed in this region, and migrated to the center of the UV‐irradiated region during its formation. When the UV‐irradiated region was displaced or its shape was changed after the formation of the sperm aster, the aster migrated to the center of the new UV‐irradiated region. The direction of the migration of the sperm aster coincided with the direction of the longest astral rays. Direct contact between astral rays and the egg surface was not essential for sperm aster migration. When a region containing both the sperm centrosome and the female pronucleus was irradiated with UV light, the female pronucleus migrated toward the center of the sperm aster after they were connected by astral rays. The migration was suppressed when UV light was shaded over the region between the aster and the female pronucleus. These results suggest that the female pronucleus migrates to the sperm aster by attractive force between them.

FERTILIZATION PROCESS IN THE HEART-URCHIN, CLYPEASTER JAPONICUS OBSERVED WITH A DIFFERENTIAL INTERFERENCE MICROSCOPE*

The observations of the fertilization process in the heart‐urchin, Clypeaster japonicus with a differential interference microscope indicate that the sperm pronucleus is carried to the center of the egg by the growth of the sperm aster as stated by Chambers (5), and that the egg pronucleus is carried to the center of the aster by a filamentous structure formed between them. The curved path of egg pronucleus in the fertilized egg is interpreted as the combination of the movement of the center of the aster and the movement of the egg pronucleus toward the center of the aster. The movement and the rotation of the sperm head result from pushing by the tail being engulfed in the egg.

Mechanical properties of the endoplasm in starfish oocytes.

Abstract The mechanical properties of the endoplasm were determined in oocytes and mature eggs of the starfish, Asterina pectinifera as follows. The cell was first deformed into a dumbbell shape by aspirating it through a circular hole of 35 or 50 μm radius formed in an agar plate of about 150 μm thickness. The movement of endoplasm in the cylindrical part of the cell was determined when a definite pressure was applied between both sides of the plate. Mechanical properties were practically represented by a viscoelastic model (fig. 5 a ) consisting of a Voigt element and a viscous element connected in series. The strain was proportional to the 0.60 ± 0.17th power of the stress in mature eggs and to the 0.76 ± 0.17th power of the stress in primary oocytes. Viscoelastic coefficients (G, η 1 and η 2 shown in fig. 5) of endoplasm changed in parallel to one another during maturation of the oocyte. They decreased with the breakdown of the germinal vesicle, increased before the extrusion of the first polar body, decreased during and after the first polar-body formation, increased before the extrusion of the second polar body, and decreased during and after the second polar-body formation.

Microinjected Polystyrene Beads Move Along Astral Rays in Sand Dollar Eggs

Movements of polystyrene beads along astral rays of the sperm aster and the mitotic aster were investigated in eggs of the sand dollars, Clypeaster japonicus and Scaphechinus mirabilis. Polystyrene beads injected into the unfertilized egg were at a standstill in the protoplasm. After fertilization, these beads exhibited movements toward the center of the sperm aster along the rays, and finally gathered around the astral center. They were distributed in blastomeres together with the mitotic centers during successive cleavages. When injected into eggs during mitosis, beads moved to the centers of the mitotic asters along astral rays. The injected beads did not move when the aster was disorganized by treatment with Colcemid, and moved when it formed after UV‐irradiation. These results indicate that microtubules of astral rays are essential to the movement of polystyrene beads. The movement of small polystyrene beads (0.2–0.3 μm in diameter) resembled the saltatory movement of endogenous cytoplasmic granules, and the movement of large beads (ca. 1 μm in diameter) resembled the female pronuclear migration. All of these movements observed in fertilized eggs were demonstrated to be microtubule‐dependent, perhaps sharing the same basic mechanisms.

Protoplasmic movement during polar-body formation in starfish oocytes

Abstract The movement of protoplasm during the formation of the polar body in starfish oocytes was analysed by tracing the movements of granules in the cell as index markers, which were recorded by 16 mm cinemicrography. Preparatory to the formation of the polar body, the cell surface expanded at the animal pole and contracted at the vegetal pole, accompanying the movement of endoplasm from the vegetal pole toward the animal pole. With the extrusion of the polar body, the cell surface around the extruded region contracted and the surface at the vegetal pole expanded, accompanying the endoplasmic movement from the animal pole toward the vegetal pole. After extrusion of the polar body, the cell surface at the animal pole expanded, accompanying the endoplasmic movement toward the animal pole in the animal hemisphere. From the movements of the surface and the endoplasm mentioned above and the change in cell shape during polar-body formation, it was concluded that the extrusion of the polar body is caused by the flow of protoplasm into the cell region at the animal pole by internal pressure of the cell through a ring-shaped region of the cortex which actively contracts to form the furrow neck.

The Role of Intracellular pH in Fertilization of Sand Dollar Eggs Analyzed by Microinjection Method

The change in intracellular pH (pHi) upon fertilization and the effects of changing the pHi by microinjection of pH buffers were investigated in the eggs of the sand dollar, Clypeaster japonicus. The pHi was determined by the tint of a pH indicator, phenol red, microinjected into eggs. The pHi ranged from 6.5 to 6.75 in unfertilized eggs and it rose by 0.4 to 0.5 unit within 3 min upon fertilization. The elevated pHi ranging from 7.0 to 7.25 was maintained at least until the first cleavage. As reported in eggs of other species of sea urchin (1–4), development of fertilized eggs which had been transferred to Na‐free sea water immediately after insemination was arrested and the pHi did not rise remaining at the level of unfertilized eggs. Development was initiated in eggs arrested in Na‐free sea water when the pHi was elevated up to the level of fertilized eggs, i.e. 7.0 to 7.25, by microinjecting 1 M HEPES (N‐2‐hydroxyethylpiperazine‐N′‐2‐ethanesulfonic acid)‐KOH buffer at pH 8.0. By microinjection of pH 7.5 buffer, some eggs started development though none of them underwent cleavage. By microinjection of pH 7.0 or pH 6.5 buffer, development was not initiated. The initiation of development depended on the pH value of microinjected pH buffer, and in consequence, on the final pHi. The elongation of microvilli which had been arrested in eggs in Na‐free sea water was also induced by microinjection of pH 8.0 or 7.5 buffer.

Effects of intracellular ph on the mitotic apparatus and mitotic stage in the sand dollar egg

The effect of change in intracellular pH (pHi) on mitosis was investigated in the sand dollar egg. The pHi in the fertilized egg of Scaphechinus mirabilis and Clypeaster japonicus, which was 7.34 and 7.31, respectively, changed by means of treating the egg at nuclear envelope breakdown with sea water containing acetate and/or ammonia at various values of pH. The mitotic apparatus at pHi 6.70 became larger than that of normal fertilized eggs; that is, the mitotic spindle had the maximal size, especially in length at pHi 6.70. The spindle length linearly decreased when pHi increased from 6.70 to 7.84. By polarization microscopy, the increase in birefringence retardation was detected at slightly acidic pHi, suggesting that the increase in size of the spindle is caused by the increase in the amount of microtubules in the spindle. At pHi 6.30, the organization of the mitotic apparatus was inhibited. Furthermore, slightly acidic pHi caused cleavage retardation or inhibition. By counting the number of the eggs at various mitotic stages with time after treating them with the media, it is found that metaphase was persistent and most of the S. mirabilis eggs were arrested at metaphase under the condition of pHi 6.70. It is concluded that at slightly acidic pH, the microtubules in the spindle are stabilized and more microtubules assembled than those in the normal eggs.

The tension at the top of the animal pole decreases during meiotic cell division.

Meiotic maturation is essential for the reproduction procedure of many animals. During this process an oocyte produces a large egg cell and tiny polar bodies by highly asymmetric division. In this study, to fully understand the sophisticated spatiotemporal regulation of accurate oocyte meiotic division, we focused on the global and local changes in the tension at the surface of the starfish (Asterina pectinifera) oocyte in relation to the surface actin remodeling. Before the onset of the bulge formation, the tension at the animal pole globally decreased, and started to increase after the onset of the bulge formation. Locally, at the onset of the bulge formation, tension at the top of the animal pole began to decrease, whereas that at the base of the bulge remarkably increased. As the bulge grew, the tension at the base of the bulge additionally increased. Such a change in the tension at the surface was similar to the changing pattern of actin distribution. Therefore, meiotic cell division was initiated by the bulging of the cortex, which had been weakened by actin reduction, and was followed by contraction at the base of the bulge, which had been reinforced by actin accumulation. The force generation system is assumed to allow the meiotic apparatus to move just under the membrane in the small polar body. Furthermore, a detailed comparison of the tension at the surface and the cortical actin distribution indicated another sophisticated feature, namely that the contraction at the base of the bulge was more vigorous than was presumed based on the actin distribution. These features of the force generation system will ensure the precise chromosome segregation necessary to produce a normal ovum with high accuracy in the meiotic maturation.

Measurement of the intracellular pH threshold for sperm aster formation in sea urchin eggs

In the fertilization of sea urchin eggs, intracellular [Ca2+] (Cai) increases transiently and intracellular pH (pHi) elevates accordingly. Unlinking these two activating factors experimentally, the requirement of the increase in pHi for sperm aster formation in the sea urchin, Clypeaster japonicus, was investigated. When the eggs were injected with an EGTA or BAPTA solution, they incorporated sperm but did not organize the sperm aster. Using these sperm‐incorporated eggs under the condition that an increase in Cai was blocked, pHi was regulated by two methods: (i) perfusing ammonium acetate‐containing seawater; and (ii) injecting pH buffer solutions of various pH values. By either of the two methods, the sperm aster formed at pHi 7.0 or more and functioned in female pronuclear migration when the sperm aster reached the female pronucleus. Hence, the step of the transient increase in Cai at fertilization can be bypassed. In contrast, a pHi increase is indispensably required for sperm aster formation in sea urchin eggs. Moreover, under the condition that there was the transient increase in Cai, the threshold pHi value for sperm aster formation was pHi 7.0 or more. Consequently, whether a Cai increase on fertilization occurs or not, the threshold pHi value for sperm aster formation is constant in sea urchin eggs.

Sperm-egg fusion in the sea urchin is blocked in Mg(2+)-free seawater.

Magnesium ions as well as calcium ions are required for successful fertilisation in sea urchins. In the absence of Mg2+ spermatozoa attached to the egg plasma membrane, their acrosomal processes passing through the vitelline envelope, but could not enter the egg cytoplasm (Sano et al., Dev. Growth Differ. 22, 531-41, 1980). Such an individual spermatozoon was observed microscopically to resume entry into the egg immediately after the addition of a sufficient amount of Mg2+ to the surrounding medium. Neither any change in membrane potential nor an increase in intracellular Ca2+ concentration of the egg was observed after insemination in the absence of Mg2+, although both could be observed after the addition of Mg2+. The sperm heads did not show fluorescence when attached to the surface of an egg previously microinjected with mithramycin A in Mg-free seawater, indicating that there was no connection between the sperm and the egg. Therefore, occurrence of fertilisation potential must be a post-fusional event. These results suggest that Mg2+ are indispensable for fusion between the sperm acrosomal membrane and the egg plasma membrane.