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.

Developmental Timing in Fertilized Merogones of the Sand Dollar, Clypeaster japonicus: (merogone/cleavage/sea urchin/DNA synthesis/mitosis).

Pairs of nucleate and non-nucleate fragments prepared by manually bisecting unfertilized eggs of Clypeaster japonicus were inseminated to obtain a pair of egg fragments, one with a diploid nucleus (zygote) and the other with a male pronucleus (merogone). Haploid male pronuclei in the merogone always entered the first mitosis (NEB) later than the zygote partner. The delay in NEB ranged from 6 to 24 min in different pairs. The interval from the first NEB to formations of the karyomere and the cleavage furrow, and the durations of the second, third and fourth cell cycles were identical in the zygote-merogone pairs. Observations of Colcemid-treated eggs suggested that the duration of the first DNA synthetic period was prolonged in the merogone. From this observation of wide variation in the time of the first mitosis, in contrast to constant durations of subsequent mitotic intervals, we suppose that some critical event triggers the transition of development for the start of well-ordered sequences in the mitotic cycle in sea urchin development.

Regional Response to Cytochalasin B of the Equatorial Cell Cortex in Sea-Urchin Eggs during the First Mitosis: (surface architecture/cortical microfilaments/cytochalasin B/sea-urchin egg/first mitosis).

Sea-urchin eggs treated with cytochalasin B (CB) during the first mitosis were examined for topography of the cell surface and the cortical microfilaments. Scanning electron microscopy revealed microvillar elongation and clumping on the whole cell surface of interphase and prophase eggs, but such changes were restricted to the equatorial surface in eggs treated with CB after metaphase up to the end of telophase. The clumping occurred also all over the cell surface of binuclear eggs after prolonged treatment up to the following interphase. These indicate localization of a target structure(s) of CB during mid- and late mitotic stages. Extraction with Triton X-100 allowed detection of CB-induced alterations in cortical microfilaments: The microvillar microfilaments were transformed into short filaments with random orientation. The submembranous meshwork and the contractile ring microfilaments were transformed into a network of shorter filaments. The prolonged CB treatment led to incorporation of the network microfilaments into the basal portions of the microvillar clumps. Thus, CB application during mid- and late mitotic stages induced (a) local microvillar changes and (b) alterations in the meshwork and the contractile ring. The present results suggest that the microvillar changes are caused by alterations of underlying cortical microfilamentous structures.

Periodicity of Cytoplasmic Cycle in Nonnucleate Fragments of Sea Urchin and Starfish Eggs*: (cortical tension/sea urchin/starfish/cytoplasmic cycle).

Fertilized eggs of the sea urchin, Hemicentrotus pulcherrimus and the starfish, Asterina pectinifera were separated by manual bisection to obtain pairs of nucleate and non-nucleate fragments. Simultaneous observation on the pair revealed that the cyclic change in the cortical tension in the non-nucleate fragment was definitely prolonged than the cleavage interval of nucleate partner by about 30%. Activated non-nucleate cytoplasmic fragments derived from the unfertilized eggs and colchicine treated whole eggs of H. pulcherrimus still showed the same degree of prolongation.

Ultrastructural Basis of the Tension Increase in Sea-Urchin Eggs Prior to Cytokinesis: (microfilament/sea-urchin egg/tension).

Cortices of sea-urchin eggs were studied by electron microscopy to identify the structure responsible for the rise in tension at the egg surface prior to cleavage. During anaphase the tension increased and fine filaments of 70-90 Å in diameter appeared in the cell cortex forming a thin mesh-work beneath the cell membrane. The meshwork spread all around the egg cortex without reference to the mitotic axis and the number of filaments seemed to increase up to telophase. Immediately before appearance of the cleavage furrow, the meshwork in the anticipated furrow region became dense. As the furrow appeared the tension began to decrease and the meshwork disappeared. In the progressing furrow region fine filaments of the same size as that of the meshwork-filament were oriented in a bundle to form a contractile ring. Treatment with cytochalasin B suppressed both the tension increase and the formation of the filamentous meshwork. These results suggest that the component filament of the meshwork is an actin microfilament, and that the tension increase at anaphase is due to formation of a meshwork of actin microfilaments from which a contractile ring is subsequently derived at late telophase.

TENSION AT THE HIGHLY STRETCHED SURFACE OF SEA-URCHIN EGGS.

Unfertilized eggs of the sea urchin, Paracentrotus lividus, were placed between two parallel plates and flattened by a definite force to 20% of their original diameter, with two-fold increase in their surface area. The resulting tension at their surface was calculated from the relation of force and deformation. In spite of this extensive stretching, the tension was found to be not more than 0.2 dyne/cm, while under conditions involving mild stretching (3%) the tension still amounted to 0.12 dyne/cm. These results do not support Mela's theory (7, 8), which predicts a transition of the mechanical properties of the egg surface from a 'subelastic' to 'elastic' state when the surface is stretched to beyond 34% of its initial area.

MARKED DECREASE IN THE RIGIDITY OF STARFISH OOCYTES INDUCED BY 1-METHYLADENINE1.

Extreme rigidity of immature starfish oocytes as measured by compression method was found to decline during the early phase of their maturation when induced by 1-methyladenine (1-MeAde). The onset of this decrease in stiffness occurred within 5 to 9 min of 1-MeAde treatment, well before the breakdown of the germinal vesicle, progressively declining to reach a minimum stiffness after 20 min. Dithiothreitol, known as an artificial maturation-inducing agent, caused a similar change. The stiffness is thus expected to serve as a quantitative indicator of the early process of cytoplasmic events, which would induce the breakdown of the germinal vesicle. Cytochalasin B (3 µg/ml) also reduced the stiffness, but unlike the former two agents, the effect was reversible, and did not interfere with the process of maturation. Due to the effect of cytochalasin B, it became possible to enucleate immature oocytes by centrifugal force. Non-nucleate fragments thus obtained still maintained their marked stiffness, which was decreased by the action of 1-MeAde, with a time-course similar to that of intact oocytes.

PERIODIC CHANGE IN THE TENSION AT THE SURFACE OF ACTIVATED NON-NUCLEATE FRAGMENTS OF SEA-URCHIN EGGS.

Unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, were separated into two fragments by centrifugal force. The enucleate fragment (merogone) was subsequently activated by treating it with butyric acid and the tension at the surface was continuously measured by a compression method. The activated merogone was found to exhibit cyclic changes in tension, with a temporal pattern very similar to that of the changes accompanying the division cycle of normally fertilized eggs. This indicates quantitatively the presence in the cytoplasm of some periodic activity which can be triggered without nuclear control. Further, a periodic thickening of the intrahyaloplasmic space of the activated merogone, as noted by Kojima (14), was confirmed on the basis of extended observation.

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. A cyclic change in both the protein-SH and the tension at the surface was found to accompany the division cycle, including the first and second meiotic divisions. It is concluded that the unequal meiotic divisions share the same character with the equal divisions of cleavage, with respect to changes both in the protein-SH and the tension at the surface.

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.