RESUMEN
To investigate whether or not causal relationship exists between the increase in intracellular Ca2+ and other cortical reactions at fertilization in the medaka, Oryzias latipes, intracellular Ca2+ was determined from luminescence of aequorin previously microinjected into cortical cytoplasm in acetone-treated eggs, when they were inseminated or activated by microinjection of Ca2+ . Neither an increase in cytoplasmic calcium nor exocytosis of cortical alveoli occurred in eggs treated with acetone, though other events of fertilization i.e. completion of meiosis, fusion of pronuclei, and accumulation of cortical cytoplasm with intact cortical alveoli in the animal pole region were observed in normal time sequence in these eggs. When denuded eggs were treated with acetone, contraction of the egg and slow resumption of meiosis (extrusion of polar body) were observed without insemination. When denuded eggs were inseminated immediately after acetone-treatment, the number of spermatozoa that penetrated into the egg was greater in the animal hemisphere than in the vegetal hemisphere. These results may indicate that acetone inactivates the egg plasma membrane or its adjacent cortical cytoplasm so that it cannot participate in a propagative increase in intracellular Ca2+ and exocytosis, while it also induces cytoplasmic activation leading to egg contraction, resumption of meiosis and formation of pronuclei. The present results suggest that sperm penetration, resumption of meiosis and ooplasmic segregation are regulated separately from the release of intracellular Ca2+ and exocytosis.
RESUMEN
A transient increase in intracellular Ca2+ 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 Ca2+ transient over the entire cell though it did not induce the germinal vesicle breakdown (GVBD). Ten minutes application of 1-MeAde induced a similar Ca2+ transient followed by GVBD. Even when the transient increase of Ca2+ was inhibited by injecting EGTA into the oocyte, 1-MeAde treatment for a long period induced GVBD. These facts indicate that the Ca2+ increase is neither necessary nor sufficient for maturation of the starfish oocyte. When the oocyte, which had been treated with 1-MeAde for 1 min at a limited area around the animal pole, was treated again with 1-MeAde for 10 min starting about 15 min after the first treatment, a Ca2+ transient similar to the first one was induced and was followed by GVBD. By contrast, in the oocyte treated with 1-MeAde at an area around the vegetal pole, neither Ca2+ transient nor GVBD was induced by the second treatment with 1-MeAde. These results indicate a difference in responsiveness to the hormone between the animal hemisphere and the vegetal hemisphere of the oocyte.
RESUMEN
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.
RESUMEN
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. In fertilized eggs, the movement of the particle by the centrifugal acceleration was impeded by the structures of the sperm aster and the cleavage diaster. The apparent viscosity of the cytoplasm in fertilized eggs changed in parallel to the development of the sperm aster and the mitotic diaster in the cell. These results indicate that the asters are really rigid structures in the cell as previously shown by the magnetic particle method (8).
RESUMEN
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. Histological examinations of fertilized and activated eggs revealed that the nucleus was in most cases positioned close to the initiation site of SCWs under various experimental conditions. It is suggested from these results that the egg cytoplasm has an intrinsic capability of causing the surface to generate SCWs, and that the nucleus is generally involved in localizing the initiation site of SCWs in fertilized or activated Xenopus eggs. A possible mechanism for localizing the initiation site of SCWs in Xenopus eggs is proposed.
RESUMEN
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 ß-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 1.3 pl, the majority of the injected embryos underwent at least one further cleavage. Expression of injected genes, which occurred transiently after injection, required the promoter sequences but without much distinction of the source of enhancer/promoter complexes. This result was in a sharp contrast to transfection of mouse cell lines where the recombinant genes were variably expressed reflecting differential enhancer effects. By injection at the 1-cell stage, expression of injected genes was low while the expression by injection at the 2-cell or later stages was several fold higher, which may correlate with the fact that most zygotic gene expression begins after the 2-cell stage. The low expression at the 1-cell stage was augmented by the conditions causing clea***age arrest such as inhibition of DNA synthesis with aphidicolin.
RESUMEN
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. The cells carrying the gene contributed to virtually any kind of tissue and their distribution varied from one tissue to another. For instance, in the neural retina, gene-positive cells formed columns several cells wide, indicating that migration of the cells derived from the founder cells is mainly along the radial axis. However, in other tissues we examined, clusters of the marked cells were less obvious, indicating the occurrence of extensive cell mixing during histogenesis. Thus, mosaic analysis of cell lineage in mouse ontogeny appears meaningful in early developmental stages or when clonal outgrowth takes place in a tissue.
RESUMEN
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.
RESUMEN
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. A Ca2+ wave was also demonstrated in sand dollar eggs, although due to their smaller size the phenomenon was not as clear as in medaka eggs.
RESUMEN
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.
RESUMEN
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 stiffness of the cell surface around the forming polar body increased during the formation of the polar body and remained at a high level after the polar body formation. It seems that the stiffness difference responsible for the formation of the contractile ring develops simultaneously with rather than prior to the formation of the cleavage furrow.
RESUMEN
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. The birefringence of the spindle and asters was not reduced even for the periods of several hours. The incorporation of heterogeneous tubulin into the isolated anaphase MAs was demonstrated by augmentation of the birefringence at the interzonal region as well as half spindles accompanied by enlargement of spindle and asters. In the anaphase MAs isolated in the absence of brain tubulin, chromosomes moved a short distance toward the poles upon addition of ATP, Mg2+ and 0.5 mg/ml tubulin. When the MAs were isolated in the presence of 0.5 mg/ml tubulin, the chromosomes moved in a more regular fashion to half the way to the poles accompanied by an increase in spindle length by 10 to 15%. GTP could not be substituted for ATP for inducing the motion. The chromosome motion of the isolated anaphase spindle was less significant than that of the isolated MA. Increasing tubulin concentration to 3 mg/ml, the chromosomes in the isolated MA separated at random by an unusual growth of the spindle. The stretch of the interzonal region by incorporating heterogeneous tubulin seemed to push the chromosomes apart abnormally. It was suggested that brain tubulin in a range of 0.5 mg/ml supports a tubulin-MA microtubule equilibrium favoring more regular motion of chromosomes in vitro.