Mitsuki Yoneda

PERIODIC CHANGES IN THE CONTENT OF PROTEIN‐BOUND SULFHYDRYL GROUPS AND TENSION AT THE SURFACE OF STARFISH OOCYTES IN CORRELATION WITH THE MEIOTIC DIVISION CYCLE*

The sulfhydryl content of protein and the tension at the surface were measured for starfish oocytes from the first meiotic division to the cleavage stage.

Centrosome destined to decay in starfish oocytes

In contrast to the somatic cell cycle, duplication of the centrioles does not occur in the second meiotic cycle. Previous studies have revealed that in starfish each of the two centrosomes in fully-grown immature oocytes consists of two centrioles with different destinies: one survives and retains its reproductive capacity, and the other is lost after completion of meiosis. In this study, we investigated whether this heterogeneity of the meiotic centrioles is already determined before the re-initiation of meiosis. We prepared a small fragment of immature oocyte containing the four centrioles and fused it electrically with a mature egg in order to transfer two sets of the premeiotic centrioles into the mature cytoplasm. Two asters were present in this conjugate, and in each of them only a single centriole was detected by electron microscopy. In the first mitosis of the conjugate artificially activated without sperm, two division poles formed, each of which doubled in each subsequent round of mitosis. These results indicate that only two of the four premeiotic centrioles survived in the mature cytoplasm and that they retained their reproductive capacity, which suggests that the heterogeneity of the maternal centrioles is determined well before re-initiation of meiosis, and that some factor in the mature cytoplasm is responsible for suppressing the reproductive capacity of the centrioles destined to decay.

Nucleus: cell volume ratio directs the timing of the increase in blastomere adhesiveness in starfish embryos.

Blastomeres of starfish embryos begin to increase in adhesiveness after the eighth cleavage and form a monolayered hollow blastula. To investigate factors that affect the timing of the adhesiveness increase, we changed the volume of the cytoplasm or the ploidy of embryos and examined the morphologic changes in the descendent blastomeres during early cleavage stages. In parthenogenetic embryos, in which the ploidy is doubled, the timing of the increase in adhesiveness was accelerated by one cell cycle. In contrast, the timing was delayed by approximately one cell cycle in a large‐sized embryo formed by the fusion of an egg and a non‐nucleate egg fragment. These two sets of observations are in accord with the expectation from the classical concept that the DNA : cytoplasmic ratio may direct the timing of events in early development. However, observations of small‐sized embryos with a reduced amount of cytoplasm were contradictory to the expectation based on the DNA : cytoplasmic ratio; the timing of the increase in adhesiveness in half‐sized embryos was almost the same as in control embryos and the timing was delayed by only one cell cycle in quarter‐sized embryos. Measurement of the diameters of nuclei showed that the size of nuclei was variable, depending on the stage of development, the volume of cytoplasm and ploidy. We calculated a volume ratio of nucleus to cytoplasm (N : C volume ratio) for tetraploid, large‐, half‐ and quarter‐sized embryos. We found that the embryonic cells begin to adhere always when their N : C volume ratio reaches 0.06. A plausible model for the cellular timing mechanism of cell contact is proposed..

TEMPERATURE‐DEPENDENCE OF THE TENSION AT THE SURFACE OF SEA‐URCHIN EGGS*

Tension at the surface of unfertilized sea‐urchin eggs was measured at various temperatures by compression method. It was confirmed that the tension definitely decreases with a rise in temperature. This indicates that (1) the tension at the surface as determined with the compression method is due solely to the plasma membrane, and (2) the membrane is fluid in nature.

Electric fusion of unfertilized starfish oocytes

An electric power device to fuse starfish oocytes was constructed. The outputs were supplied to a pair of parallel platinum wires in a solution of mannitol to which small amounts of salts were added. Oocytes of the starfish Asterina pectinifera, deprived of the vitelline envelope, were fused by several repetitions of a combination of 2.5 MHz AC field for a few seconds and a 50 μs DC pulse. Observation of fusing pairs of immature oocytes revealed that: (i) the oocyte placed near to the cathode forms a bulge at the surface facing the anode when subjected to DC pulses and, with subsequent DC pulses, a similar bulge is formed on another oocyte; and (ii) the pair of bulges then fuse together leading to fusion of the main body of the two oocytes. The conjugate of maturing oocytes soon became a single sphere, usually within 10 min, but this process toward spherical form paused when the oocyte was extruding its polar bodies. The conjugate of immature oocytes took 1 h to become a single sphere. The fusion did not disturb the progress of meiotic events, but electric pulses at an intensity suitable for the fusion often activated maturing oocytes and mature eggs.

Heteroplasmic conjugates formed by the fusion of starfish oocyte pairs with a 12 minute time difference in the maturation phase

Two starfish oocytes with a 12 min time difference in the maturation phase were fused together with electric pulses to make a heteroplasmic conjugate. The starfish used were Asterina pectinifera. The emergence of the first meiotic spindle and the extrusion of the polar bodies in the conjugate were timed. Under polarization microscopy two meiotic spindles emerged with a time difference of 10–11 min, which is close to the time difference in the maturation phase between the original oocytes before fusion. In contrast, subsequent formation of the first two polar bodies occurred successively with a short time lag of 1–3 min between them. Times for the formation of both polar bodies were midway between the anticipated times for polar body formation in respective non‐fused control oocytes. Thus, in one nucleus the meiotic division was delayed, while in another nucleus it was accelerated, in a single heteroplasmic conjugate. These two sets of observations indicate the presence of a certain control system that regulates progression of the cell cycle at a point during the period from the entry into metaphase through to late anaphase of meiosis I in starfish oocytes. This type of cell cycle control in starfish oocytes is obviously distinct from the currently accepted view of the cell cycle control by the spindle assembly checkpoint that monitors unattached kinetochores of mitotic chromosomes.