Yukio Hiramoto

Activation of sea urchin eggs by microinjection of calcium buffers.

Abstract The effects of changing the intracellular Ca 2+ concentration on unfertilized sea urchin eggs were investigated by microinjecting calcium buffers, i.e., aqueous solutions for stabilizing concentrations of free calcium ions (Ca 2+ ) containing calcium salt and calcium-chelating substances (ethyleneglycol bis (β-aminoethylether)- N,N′ -tetraacetic acid (EGTA) or N -hydroxyethyl-ethylenediaminetriacetic acid (HEDTA)) at various ratios. When the intracellular Ca 2+ concentration was raised to more than 0.2 μM, the elevation of fertilization membrane was observed in more than 50% of eggs in normal sea water. The threshold Ca 2+ concentration inducing membrane-elevation was 0.5 μM in eggs in Ca 2+ -free sea water. Activation accompanying the formation of a monaster was induced by raising the intracellular Ca 2+ concentration to 3–4 times the above threshold levels in both eggs in normal sea water and eggs in Ca 2+ -free sea water. Eggs were not fertilized when the intracellular Ca 2+ concentration had been clamped below 0.1 μM by microinjecting calcium buffers before insemination. When the egg was put into sea water containing 5 mM procaine, the threshold Ca 2+ concentration for inducing the membrane-elevation increased. It was concluded that the rise of Ca 2+ concentration in the cytoplasm to a definite level is the primary cause of the activation of the sea urchin egg at fertilization.

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.

The Wave Pattern of Free Calcium Release Upon Fertilization in Medaka and Sand Dollar Eggs

A transient rise in the concentration of Ca2+ 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 Ca2+ 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. The peak value of the aequorin luminescence, and therefore the peak value of the Ca2+ transient, was generally higher at the site of sperm entry than in other regions. The peak values of the luminescence (and therefore of the Ca2+ concentration in the cortex) remained fairly constant during propagation of the wave. Microinjection of Ca2+ into the cortex also induced a Ca2+ wave. When the egg was stimulated by microinjection of Ca2+ at the equatorial region, the Ca2+ wave was propagated at a fairly constant speed over the egg surface, except at the region near the vegetal pole where the wave was retarded. Simultaneous recording of the Ca2+ wave and the wave of cortical change (breakdown of cortical alveoli) in eggs during fertilization revealed that the Ca2+ wave preceded the wave of cortical change.

MECHANICAL PROPERTIES OF THE CELL SURFACE IN STARFISH EGGS

The mechanical properties of the cell surface of the starfish egg at various stages of maturation have been investigated using the cell elastimeter. When constant negative pressure was applied to a part of the cell with a micropipette closely in contact with it, it bulged out, and the bulge rapidly increased at first and then gradually reached a steady value within one min. The relation between the deformation of the cell surface (i.e. degree of bulging) and applied negative pressure was almost linear in both oocytes at the germinal vesicle stage and mature eggs. The surface force and the elasticity value: i.e., the product of the elastic modulus of the surface membrane (layer) and its thickness, were determined from the relation between the deformation and the negative pressure. The elasticity value was about 5 times the surface force in both oocytes at the germinal vesicle stage and mature eggs. When maturation of the oocyte was induced by 1‐methyladenine, the stiffness of the cell surface decreased shortly before the breakdown of the germinal vesicle. The stiffness transiently increased at the time of formation of the first and second polar‐bodies.

Specific expression of the chicken δ-crystallin gene in the lens and the pyramidal neurons of the piriform cortex in transgenic mice☆

Two transgenic mice, 5-8 and 7-5, carrying the chicken delta-crystallin gene were produced by microinjecting cloned genes into male pronuclei. The mice were analyzed at 8 weeks of age with respect to gene integration and expression by means of blotting techniques and immunohistochemistry. Southern blot analysis indicated that both mice carried, on average, 50 copies of intact delta-crystallin gene per cell. Histological analysis of the mice using DNA-DNA in situ hybridization indicated that mouse 5-8 carried the delta-crystallin gene in every cell while mouse 7-5 was mosaic, with 20-40% of the cells of various tissues carrying the gene. Western blot analysis indicated that in both mice delta-crystallin is expressed in the lens and the cerebrum, but not in any other tissue examined. Immunohistological analysis revealed that, in the cerebrum of the mice, delta-crystallin was expressed specifically in pyramidal neurons located in layer IIb of the anterior piriform cortex. Thus, our results with transgenic mice not only demonstrate the primary specificity of delta-crystallin gene expression in authentic lens tissue, but reveal the unexpected specificity of this chicken gene in the central nervous system of the mouse.

Time Sequence of Early Events in Fertilization in the 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 × 108/ml at 23°–25°C). The mean time from sperm attachment until cessation of its movement on the egg surface was 4 ± 1 sec with a range from 1 to 9 sec. Small cortical alveoli at the animal pole region within 15 μm of the sperm attachment point began to undergo exocytosis 9 ± 0.3 sec (range 5–16 sec) after sperm attachment. The velocity at which the exocytosis wave propagated increased from the earliest initiation point of exocytosis up to the 100 μm area, and became constant at about 12 μm/sec from 100 μm to 500 μm from the sperm attachment point. The present results suggest that at the time of fertilization in the fish egg, exocytosis of small cortical alveoli in the area about 15 μm away from the sperm attachment point occurs simultaneously.

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.

Introduction and Expression of Recombinant β-Galactosidase Genes in Cleavage Stage Mouse Embryos

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.

Cytoplasmic Ca2+ release induced by microinjection of Ca2+ and effects of microinjected divalent cations on Ca2+ sequestration and exocytosis of cortical alveoli in the medaka egg

Intracellular release of Ca2+ by microinjection of Ca2+ was analyzed by measuring the luminescence of aequorin loaded in eggs of the medaka (Oryzias latipes). Microinjection of Ca2+ into the cortical cytoplasm induced propagative waves of cytoplasmic Ca2+ release and exocytosis of cortical alveoli initiated at the injection point. The Ca2+ wave was initiated with a time lag after some was sequestered at the region of the microinjection. Microinjection of Mg2+ or Mn2+ failed to trigger Ca2+ release and exocytosis. When the aequorin-loaded eggs were inseminated after microinjection of Mg2+, Mn2+, or Co2+ into a restricted region of the vegetal hemisphere, the wave of Ca release was propagated through the injected region toward the vegetal pole, but neither Ca sequestration (fall in Ca-aequorin luminescence) nor exocytosis occurred at the area of cortex where the eggs were injected with these divalent cations. These results suggest that a significant period is required to induce Ca2+ release from cytoplasmic stores by the increased Ca2+ concentration and that both the phenomena of Ca2+ release and Ca sequestration are involved in the process of exocytosis.

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.