Fertilization Reaction without Changes in Intracellular Ca in Medaka Eggs-An Experiment with Acetone-Treated Eggs.

To investigate whether or not causal relationship exists between the increase in intracellular Ca and other cortical reactions at fertilization in the medaka, Oryzias latipes, intracellular Ca was determined from luminescence of aequorin previously microinjected into cortical cytoplasm in acetone-treated eggs, when they were inseminated or activated by microinjection of Ca . Neither an increase in cytoplasmic calcium nor exocytosis of cortical alveoli occurred in eggs treated with acetone, though other events of fertilization i.e.

Change in Intracellular Calcium Ions upon Maturation in Starfish Oocytes: (aequorin/calcium/oocyte maturation/1-methyladenine/starfish).

A transient increase in intracellular Ca upon maturation in starfish oocyte was revealed by light emission of aequorin microinjected into the cell. One minute application of 1-methyladenine (1-MeAde) to a limited area of the oocyte surface was sufficient to induce the Ca transient over the entire cell though it did not induce the germinal vesicle breakdown (GVBD). Ten minutes application of 1-MeAde induced a similar Ca transient followed by GVBD.

Time Sequence of Early Events in Fertilization in the Medaka Egg: (micropyle/exocytosis/sperm movement/calcium release/medaka egg).

The time sequence of early events in fertilization was examined in eggs of the medaka Oryzias latipes. The mean time after insemination required for sperm attachment to the egg surface through the micropyle depended on sperm concentrations. It was 3 ± 1 sec with a range from 1 to 6 sec after insemination when concentration of spermatozoa was high (about 2 × 10 /ml at 23°-25°C).

The Mechanical Structure of the Cytoplasm of the Echinoderm Egg Determined by "Gold Particle Method" using a Centrifuge Microscope: (gold particle method/mechanical structure/cytoplasm/echinoderm eggs/cell division).

A method was developed to investigate the mechanical structure of the cytoplasm based on the movement of an intracellular gold particle subjected to centrifugal acceleration (the gold particle method). The movement of the particle in the cell was observed and recorded with a new centrifuge microscope of stroboscopic type (13). In eggs and oocytes of the echinoderms, Clypeaster japonicus, Asterias amurensis, and Asterina pectinifera, the particle moved in the cytoplasm by an applied centrifugal acceleration in the centrifugal direction, but the course was not exactly straight and the velocity fluctuated during the movement, suggesting the existence of a network structure in the cytoplasm.

Nuclear Involvement in Localization of the Initiation Site of Surface Contraction Waves in Xenopus Eggs: (surface contraction waves/Xenopus laevis/nucleus/colchicine/cleavage).

The initiation site of surface contraction waves (SCWs) was examined in fertilized, parthenogenetically activated and enucleated Xenopus eggs after either rotation through 90° off the vertical axis or injection of colchicine. In enucleated eggs, SCWs always started from a top site of the egg under all conditions examined. In fertilized or activated eggs, SCWs started, depending on the experimental conditions, from either the sperm entry point, the animal pole region located sideward or the top site of the egg.

Analysis of Cellular Mosaicism in a Transgenic Mouse by Histological In Situ Hybridization: (Transgenic mice/mosaics/cell lineage/in situ hybridization).

One of the transgenic mice carrying a chicken δ-crystallin gene was found to be mosaic with regard to the distribution of the exogenous gene. Taking advantage of the exogenous DNA sequences as a cell lineage marker detectable by histological in situ hybridization technique, we studied cellular mosaicism in mouse 7-5. This mouse carried the exogenous gene in 20-40% of its cells, probably reflecting chromosomal integration of the exogenous DNA which occurred in a blastomere of around the 4-cell stage.

Introduction and Expression of Recombinant β-Galactosidase Genes in Cleavage Stage Mouse Embryos: (microinjection/mouse embryo/gene expression).

In an attempt to study gene regulation in very early stages of mouse embryogenesis, we injected genes constructed by joining the coding sequence of the bacterial β-galactosidase gene to four different animal gene enhancers/promoters and to poly (A) signals, and examined the gene expression in cleavage stage embryos. With appropriate injection volumes for each embryonic stage, ranging from 0.2 to 1.

The Wave Pattern of Free Calcium Release Upon Fertilization in Medaka and Sand Dollar Eggs: (Ca /fertilization/medaka/sand dollar/aequorin).

A transient rise in the concentration of Ca in the cortex upon fertilization was demonstrated in medaka eggs injected with aequorin. Detection of the aequorin luminescence with an ultra-high sensitivity photonic microscope system revealed a wave of increased Ca concentration starting at the site of sperm entry (animal pole) and being propagated along the cortex of the egg toward the antipode. The wave traversed the entire egg surface within 2-3 min.

Microinjected Polystyrene Beads Move Along Astral Rays in Sand Dollar Eggs: (astral rays/fertilization/mitosis/microinjected polystyrene beads/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.

Analysis of the Role of Astral Rays in Pronuclear Migration in Sand Dollar Eggs by the Colcemid-UV Method: (sperm aster/pronuclear migration/sand dollar/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.

Regional Difference in Mechanical Properties of the Cell Surface in Dividing Echinoderm Eggs*: (mechanical properties/cell surface/sea urchin eggs/starfish eggs/cleavage).

Stiffness of the cell was surface was determined in fertilized sea urchin and starfish eggs by measuring the mechanical resistivity of the cell surface against negative pressure applied to a restricted part with a micropipette in contact with the cell surface at its tip (elastimetry). In both sea urchin and starfish eggs, the stiffness of the cell surface changed almost in parallel between the presumptive furrow and polar surfaces before the onset of the first cleavage, and the stiffness of the furrow surface became larger than that of the polar surface when cleavage started, although temporal changes in the stiffness were different between sea urchin and starfish eggs. The stiffness of the cell surface changed almost in parallel between the surfaces at the equator and at the animal pole in starfish eggs before the onset of polar body formation.

THE GLYCEROL-ISOLATED MITOTIC APPARATUS: A RESPONSE TO PORCINE BRAIN TUBULIN AND INDUCTION OF CHROMOSOME MOTION.

The birefringence of the MAs or spindles isolated from sea urchin eggs with the 1 M glycerol-isolation medium was stabilized when more than 0.5 mg/ml tubulin was contained in the medium. The addition of glycerol up to a final concentration of of 4 M strongly stabilized the MAs even in the absence of GTP and tubulin.