Asynchronization of Cell Division is Concurrently Related with Ciliogenesis in Sea Urchin Blastulae

During the early development of the sea urchins, Temnopleurus toreumaticus, Temnopleurus hardwickii and Hemicentrotus pulcherrimus , the division synchrony in all blastomeres lasted only until the 4th cleavage and a regional synchrony or a graded activity of cell division appeared. In the midblastula stage prior to hatching, the regional synchrony vanished simultaneously with the formation of cilia, then the division proceeded asynchronously. The analysis of cell pedigrees confirmed that a variable extension of intercleavage times occurred after the ciliogenesis. In blastomeres derived from mesomeres of T. toreumaticus embryos, the mean intercleavage time extended from 48 min of the 8th cycle (pre-ciliated) to 115 min of the 9th cycle (ciliated), and the coefficient of variation increased from 15% to 39%. We attempted a kinetic analysis of cell proliferation on the basis of the transition probability model of cell cycle control. We concluded that the minimum time required for the completion of the cell cycle was the decisive factor in the cell cycle succession of pre-ciliated blastomeres, and that a sudden and sharp decrease in the transition probability of the ciliated blastomeres probably interpreted the abrupt slowing and asynchronization of the cleavage cycle at the time of ciliogenesis.     

ANALYSIS OF THE VARIABILITY IN CLEAVAGE TIMES AND DEMONSTRATION OF A MITOTIC GRADIENT DURING THE CLEAVAGE STAGES OF NOTHOBRANCHIUS GUENTHERI

Living embryos of the annual cyprinodont fish Nothobranchius guentheri were observed under the microscope. Detailed records were made of the time of cell division, disappearance of the nucleus and of the position of each cell within the blastoderm up to and including the sixth cleavage. Combination of these data revealed the presence of a mitotic gradient, a cell division gradient and a gradient of cell cycle duration in the 8-cell, 16-cell and 32-cell stage. Comparison of the variabilities in the duration of the interphase and mitosis reveals that differences between sister cell intercleavage times in the 8-, 16- and 32-cell stage are, for the most part, due to the variability in the duration of the mitotic process. It is concluded that the DNA-division cycle is composed of at least two parallel series of events. We found the random transition model of cell cycle control, originally based on the analysis of intermitotic times of mammalian cells in tissue culture, helpful also in analysing intercleavage time variability in embryonic cells.     

Transition of the blastomere cell cycle from cell size-independent to size-dependent control at the midblastula stage in Xenopus laevis

Dissociated animal cap blastomeres of Xenopus laevis blastulae were cultured at a low Ca level (1 microM) from 9th to 18th cell cycle at 22 +/- 1 degrees C and observed by a time-lapse video recorder. Blastomeres cleaved unequally to increase variability in cell size as cell cycles progressed, but synchronously at a constant cell cycle time of about 30 min up to the 12th cleavage in diploid cells, and up to the 13th cleavage in haploid cells, regardless of their cell sizes. Thereafter, blastomeres cleaved asynchronously at varying cell cycle times in proportion to the inverse square of their radii. The transition from the cell size-independent to -dependent cell cycles occurred at the critical cell radius, 37.5 microm for the diploid and 27.9 microm for the haploid. While the protein synthesis inhibitor, cycloheximide (CHX) lengthened cell cycle times two- to six-fold, epidermal growth factor (EGF) had no significant effect on the cell cycle. CHX-treated blastomeres synchronously cleaved at a constant cell cycle time of 60 min up to the 12th cleavage. Thereafter, cell cycle times became variable in proportion to the inverse square of radii in the presence of CHX at 0.10-0.14 microg/ml, but to the inverse cube of radii at 0.18 microg/ml. The critical cell size of CHX-treated blastomeres for the transition from cell size-independent to -dependent cell cycles remained the same as that of untreated blastomeres. Frequency distributions of cell cycle times of synchronous cell cycles were monomodal with the peak at 30 min, except for CHX-treated blastomeres with the peak at 60 min. In contrast, frequency distributions of asynchronous cell cycles were polymodal with peaks at multiples of a unit time of 30-35 min. To explain these results, we propose that blastomere cytoplasm has 30-min cycles that repeatedly produce mitosis promoting factor (MPF) in a quantity proportional to the cell surface area. MPF is neutralized when it titrates a nuclear inhibitor present in a quantity proportional to the genome size, and sequestered in the nucleus. When the total amount of MPF produced exceeds the threshold required to titrate all of the inhibitor, mitosis is initiated.     

Development of Microtubule-Dependence of the Chromosome Cycle at the Midblastula Transition in Xenopus laevis Embryos

Animal-cap cells isolated from Xenopus laevis morulae and blastulae are cultured for 2 to 6 hr in medium containing nocodazole, Colcemid or taxol, at concentrations completely inhibiting cell division. At 20°C, cells from each control embryo undergo synchronous cell cycles up to the 12th, with a period of 32 min, of which 60% represents the chromosome condensation (mitotic or M-) phase, and the average mitotic index remains near 50%. Cells treated with nocodazole, Colcemid or taxol before 12th cleavage undergo chromosome cycles with a similar period as controls, albeit without chromosome segregation, and the average mitotic index remains near 50%. From the 12th to 15th cycles, control cycles become asynchronous, their period gradually increases 2 to 3 times, and the mitotic index declines to 10%. In cells treated after 12th cleavage with taxol, the mitotic index declines, similarly to control cells. However, in nocodazole-treated cells, it increases steadily, and exceeds 70% at 2 hr of treatment, but gradually declines to 40% at 6 hr. Therefore, while inhibition of microtubule activities does not significantly alter the timing of chromosome condensation cycles during synchronous cleavage, inhibition of microtubule assembly can prolong M-phase during asynchronous cleavage after the midblastula transition.     

Structural heterogeneity in populations of the budding yeast Saccharomyces cerevisiae.

Bud scar analysis integrated with mathematical analysis of DNA and protein distributions obtained by flow microfluorometry have been used to analyze the cell cycle of the budding yeast Saccharomyces cerevisiae. In populations of this yeast growing exponentially in batch at 30 degrees C on different carbon and nitrogen sources with duplication times between 75 and 314 min, the budded period is always shorter (approximately 5 to 10 min) than the sum of the S + G2 + M + G1* phases (determined by the Fried analysis of DNA distributions), and parent cells always show a prereplicative unbudded period. The analysis of protein distributions obtained by flow microfluorometry indicates that the protein level per cell required for bud emergence increases at each new generation of parent cells, as observed previously for cell volume. A wide heterogeneity of cell populations derives from this pattern of budding, since older (and less frequent) parent cells have shorter generation times and produce larger (and with shorter cycle times) daughter cells. A possible molecular mechanism for the observed increase with genealogical age of the critical protein level required for bud emergence is discussed.     

Development of bovine embryo-derived clones after increasing rounds of nuclear recycling

This study assessed in vitro and in vivo developmental ability of bovine embryo-derived clones after one, four or seven rounds of nuclear transfer. Initial donor embryo production and all subsequent cultures were performed in vitro. Donor clonal embryo lines were vitrified and warmed either once (first generation), twice (third generation) or three times (sixth generation) before the final round of cloning. No differences were observed in fusion, cleavage and development rates to the 16-cell stage between the first six cloning generations. Likewise, neither the fusion nor cleavage rates were different between first, fourth and seventh generation clones. However, development to morulae and blastocysts decreased significantly as the number of recycling rounds increased (24.8, 15.1 and 13.6% for first, fourth and seventh generation, respectively). In addition, the proportion of blastocysts compared to morulae decreased, indicating slower developmental speed in later generation clones. After transfer of 16, 25 and 7 clones to 7, 11 and 2 recipients (first, fourth and seventh generation, respectively) initial pregnancy rates of 57, 27 and 0% were obtained. Final rates of calves to term were 25 and 4% per transferred clone for first and fourth generation clones, respectively. These results indicate greatly reduced in vitro and in vivo developmental capacity of bovine embryo-derived clones after several rounds of nuclear recycling. Whether it is caused by intrinsic factors associated with the genome modification and reprogramming as such, or by external factors such as prolonged in vitro culture period or the effects of vitrification, remains to be determined.     

Involvement of a urethane-sensitive system in timing the onset of gastrulation in Xenopus laevis embryos

This paper describes success in delaying the onset of gastrulation in Xenopus laevis embryos without damage to their subsequent development by temporarily arresting cleavage with urethane. Exposure of X. laevis embryos to 150 mM urethane before gastrulation resulted in cleavage arrest and its removal led to cleavage resumption. During cleavage arrest, cyclic activities including nuclear replication and the M-phase-promoting factor cycle continued, although their duration was lengthened to nearly 1.8-fold that of the controls. Because of a 30-min time lag from removal of urethane to resumption of cleavage, as well as the retardation of cyclic activities during cleavage arrest, the development of embryos after a 60-min exposure to urethane lagged two cell cycles behind that of control embryos. Here, the two cell cycle delay is equivalent to 50 min at 22-23 degrees C. The start of gastrulation in exposed embryos was accordingly delayed about 50 min, although the delay in mid-blastula transition was as little as 20-25 min. Consistent results were obtained in embryos exposed to urethane for 90 or 120 min and those exposed to procaine or NH4Cl for 60 min. Although these results imply that delay in the start of gastrulation in exposed embryos is ascribed simply to delay in their development raised by cleavage arrest, at the same time they suggest that the onset of gastrulation is timed by systems sensitive to urethane, procaine and NH4Cl in X. laevis embryos.     

Human lymphokine-activated killer (LAK) cells

Summary Pretreatment of peripheral blood mononuclear cells (PBMC) with 5 mMl-phenylalanine methyl ester (PheOMe) provides an efficient means to deplete monocytes. PheOMe does not affect the number of large granular lymphocytes after the pretreatment, but does inhibit natural killer cell cytotoxicity temporarily after the pretreatment. However, depletion of monocytes by PheOMe allows lymphokine-activated killer (LAK) cell generation with recombinant interleukin-2 (rIL-2) at high cell density (> 5 × 10⁶ cells/ml). The time of the PheOMe pretreatment is 40–60 min, though some effect could be observed within 15 min, and the pretreatment could be performed at room temperature. Pretreatment density of PBMC with 5 mM PheOMe could be achieved at cell density up to 3 × 10⁷ cells/ml. PheOMe-pretreated cells could be activated by rIL-2 in serumless media at high cell density. Pretreatment of PBMC with 5 mM PheOMe provides an efficient means to deplete monocytes, as compared to plastic and nylonwool adherence. LAK cell generation is similar in both methods of monocyte depletion; therefore, depletion of monocytes allows, LAK cell generation at high cell density. The PheOMe procedure provides an improved and convenient process for preparing LAK cells for adoptive immunotherapy.     

Embryonic stages from cleavage to gastrula in the loach Misgurnus anguillicaudatus

Early developmental staging from the zygote stage to the gastrula is a basic step for studying embryonic development and biotechnology. We described the early embryonic development of the loach, Misgurnus anguillicaudatus, based on morphological features and gene expression. Synchronous cleavage was repeated for 9 cycles about every 27 min at 20 degrees C after the first cleavage. After the 10th synchronous cleavage, asynchronous cleavage was observed 5.5 h post-fertilization (hpf), indicating the mid-blastula transition. The yolk syncytial layer (YSL) was formed at this time. Expressions of goosecoid and no tail were detected by whole-mount in situ hybridization from 6 hpf. This time corresponded to the late-blastula period. Thereafter, epiboly started and a blastoderm covered over the yolk cell at 8 hpf. At 10 hpf, the germ ring and the embryonic shield were formed, indicating the stage of early gastrula. Afterward, the epiboly advanced at the rate of 10% of the yolk cell each hour. The blastoderm covered the yolk cell completely at 15 hpf. The embryonic development of the loach resembled that of the zebrafish in terms of morphological change and gene expression. Therefore, it is possible that knowledge of the developmental stages of the zebrafish might be applicable to the loach.     

Heat shock dependent fluctuations of RNase activity during the cell cycle of synchronized Tetrahymena.

The total cellular acid RNase activity per milliliter of culture increases sharply following each heat shock in the cell cycle of Tetrahymena pyriformis ST synchronized with heat shocks spaced one generation time apart. Thus, the RNase activity per 10(5) cells is 24.5 units immediately after the end of the sixth heat shock, increases to 39.0 units during the following 55 minutes and decreases to 24.2 units at the start of the seventh heat shock. No change in the RNase activity occurs during the heat shock period. In logarithmically growing cells the RNase activity per 10(5) cells is 15.4 units. The heart shock stimulates the increase in the RNase activity, since no rapid increase occurs during the free running division cycle but a rapid increase occurs after an additional heat shock given at different times during the cell cycle. Inhibition of the increase in RNase activity by cycloheximide suggests that concurrent protein synthesis is required for the stimulation of the RNase activity by the heat shock treatment.     

Timing and Regulation of Nuclear and Cortical Events in the Cell Cycle of Tetrahymena pyriformis*

SYNOPSIS. Using continuous flow cultures based on the chemostat principle, we varied the cell generation times of the ciliate Tetrahymena pyriformis strain GL, from 4.9 to 22.2 hr and studied various parameters of the cell cycle at 28 C. These included: the duration of the periods required for oral morphogenesis, macronuclear division, cell division, G₁ S, and G₂. The size of individual cells was also measured. Independent of the growth rate, the period of oral morphogenesis occurred during the last 90 min of the cell cycle. In all cases macronuclear and cell divisions took place during the last part of these 90 min, and the final macronuclear separation occurred just before final cell separation. The S-period increased slightly, while the G₁ and G₂ both increased in roughly the same relative proportion to the increasing generation times. Slowly growing cells (generation time 20.5 hr) were shorter but broader and somewhat larger in volume than quickly growing cells (generation time 4.9 hr).     

Translocation of a store of maternal cytoplasmic c-myc protein into nuclei during early development.

The c-myc proto-oncogene is expressed as a maternal protein during oogenesis in Xenopus laevis, namely, in nondividing cells. A delayed translation of c-myc mRNA accumulated in early oocytes results in the accumulation of the protein during late oogenesis. The oocyte c-myc protein is unusually stable and is located in the cytoplasm, contrasting with its features in somatic cells. A mature oocyte contains a maternal c-myc protein stockpile of 4 x 10(5) to 6 x 10(5) times the level in a somatic growing cell. This level of c-myc protein is preserved only during the cleavage stage of the embryo. Fertilization triggers its rapid migration into the nuclei of the cleaving embryo and a change in the phosphorylation state of the protein. The c-myc protein content per nucleus decreases exponentially during the cleavage stage until a stoichiometric titration by the embryonic nuclei is reached during a 0.5-h period at the midblastula stage. Most of the maternal c-myc store is degraded by the gastrula stage. These observations implicate the participation of c-myc in the events linked to early embryonic development and the midblastula transition.     

Computerized video time-lapse analysis of apoptosis of REC:Myc cells X-irradiated in different phases of the cell cycle

Asynchronous rat embryo cells expressing Myc were followed in 50 fields by computerized video time lapse (CVTL) for three to four cycles before irradiation (4 Gy) and then for 6-7 days thereafter. Pedigrees were constructed for single cells that had been irradiated in different parts of the cycle, i.e. at different times after they were born. Over 95% of the cell death occurred by postmitotic apoptosis after the cells and their progeny had divided from one to six times. The duration of the process of apoptosis once it was initiated was independent of the phase in which the cell was irradiated. Cell death was defined as cessation of movement, typically 20-60 min after the cell rounded with membrane blebbing, but membrane rupture did not occur until 5 to 40 h later. The times to apoptosis and the number of divisions after irradiation were less for cells irradiated late in the cycle. Cells irradiated in G(1) phase divided one to six times and survived 40-120 h before undergoing apoptosis compared to only one to two times and 5-40 h for cells irradiated in G(2) phase. The only cells that died without dividing after irradiation were irradiated in mid to late S phase. Essentially the same results were observed for a dose of 9.5 Gy, although the progeny died sooner and after fewer divisions than after 4 Gy. Regardless of the phase in which they were irradiated, the cells underwent apoptosis from 2 to 150 h after their last division. Therefore, the postmitotic apoptosis did not occur in a predictable or programmed manner, although apoptosis was associated with lengthening of both the generation time and the duration of mitosis immediately prior to the death of the daughter cells. After the non-clonogenic cells divided and yielded progeny entering the first generation after irradiation with 4 Gy, 60% of the progeny either had micronuclei or were sisters of cells that had micronuclei, compared to none of the progeny of clonogenic cells having micronuclei in generation 1. However, another 20% of the non-clonogenic cells had progeny with micronuclei appearing first in generation 2 or 3. As a result, 80% of the non-clonogenic cells had progeny with micronuclei. Furthermore, cells with micronuclei were more likely to die during the generation in which the micronuclei were observed than cells not having micronuclei. Also, micronuclei were occasionally observed in the progeny from clonogenic cells in later generations at about the same time that lethal sectoring was observed. Thus cell death was associated with formation of micronuclei. Most importantly, cells irradiated in late S or G(2) phase were more radiosensitive than cells irradiated in G(1) phase for both loss of clonogenic survival and the time of death and number of divisions completed after irradiation. Finally, the cumulative percentage of apoptosis scored in whole populations of asynchronous or synchronous populations, without distinguishing between the progeny of individually irradiated cells, underestimates the true amount of apoptosis that occurs in cells that undergo postmitotic apoptosis after irradiation. Scoring cell death in whole populations of cells gives erroneous results since both clonogenic and non-clonogenic cells are dividing as non-clonogenic cells are undergoing apoptosis over a period of many days.     

Eclipse period during replication of plasmid R1: contributions from structural events and from the copy-number control system

The eclipse period (the time period during which a newly replicated plasmid copy is not available for a new replication) of plasmid R1 in Escherichia coli was determined with the classic Meselson-Stahl density-shift experiment. A mini-plasmid with the wild-type R1 replicon and a mutant with a thermo-inducible runaway-replication phenotype were used in this work. The eclipses of the chromosome and of the wild-type plasmid were 0.6 and 0.2 generation times, respectively, at temperatures ranging from 30 degrees C to 42 degrees C. The mutant plasmid had a similar eclipse at temperatures up to 38 degrees C. At 42 degrees C, the plasmid copy number increased rapidly because of the absence of replication control and replication reached a rate of 350-400 plasmid replications per cell and cell generation. During uncontrolled replication, the eclipse was about 3 min compared with 10 min at controlled replication (the wild-type plasmid at 42 degrees C). Hence, the copy-number control system contributed significantly to the eclipse. The eclipse in the absence of copy-number control (3 min) presumably is caused by structural requirements: the covalently closed circular plasmid DNA has to regain the right degree of superhelicity needed for initiation of replication and it takes time to assemble the initiation factors.     

Study of the Lineage and Cell Cycle of Small Micromeres in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

A study was made of 1st cell cycle of small micromeres, segregated at the 5th cleavage cycle, in the sea urchin embryos of Hemicentrotus pulcherrimus . For identification of small micromeres, the embryos were pulse labeled with 5-bromodeoxyuridine (BrdU) at the 1st cleavage. Using multiparametric microfluorometry equipped with a scanning stage (Tanaka, 1990), DNA content, extent of BrdU incorporation, protein content and the extent of ³H-thymidine labeling were measured on identical individual cells dissociated from an embryo. The findings of the present study are as follows. There is a short period of time between the telophase and onset of DNA replication. The period of DNA replication is 5 hr and after which, asynchronous mitosis takes place to produce 8 cells before hatching. The long S period is 83% the total 6 hr of the cell cycle. The rate of DNA accumulation is quite small during the initial one third of S but increases later in this phase. The degree of chromatin condensation remains high even during the S phase but it is low in large micromeres. The cell cycle may possibly be related causally to the development of small micromeres. The developmental significance of cell cycle duration, particularly that of DNA replication is discussed.     

Daunorubicin- and Ara-C-induced interphasic apoptosis of human type II leukemia cells is caspase-8-independent

Drug-induced interphasic apoptosis in human leukemia cells is mediated through intracellular signaling pathways, of which the most proximal (initiating) event remains unclear. Indeed, both early ceramide generation and procaspase-8 cleavage have been individually identified as the initial apoptotic signaling events which precede the mitochondrial control of the apoptotic execution phase in Type II cells. In order to evaluate whether or not procaspase-8 cleavage is requisite for initial ceramide generation and rapid interphasic apoptosis, we investigated the chronological ordering of early ceramide generation and caspase-8 cleavage induced by daunorubicin (DNR) and 1-beta-D-arabinofuranosylcytosine (Ara-C) in U937 cells. We further evaluated the impact of these two drugs on initial ceramide generation and apoptosis in wild-type Jurkat cells and Jurkat clones mutated for caspase-8 and Fas-associated death domain. We show that while both DNR and Ara-C similarly induced early ceramide generation (within 5-20 min) and interphasic apoptosis in all cell models, caspase-8 cleavage was only observed farther downstream (4.5 h) and only in DNR-treated cells. Furthermore, neither DNR or Ara-C induced caspase-8 activation. These results demonstrate that caspase-8 cleavage is not requisite for the drug-induced activation of the ceramide-mediated interphasic apoptotic pathway in human Type II leukemic cells.     

Phorbol esters alter cell fate during development of sea urchin embryos

Protein kinase C (PKC) has been implicated as important in controlling cell differentiation during embryonic development. We have examined the ability of 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of PKC, to alter the differentiation of cells during sea urchin development. Addition of TPA to embryos for 10-15 min during early cleavage caused dramatic changes in their development during gastrulation. Using tissue-specific antibodies, we have shown that TPA causes the number of cells that differentiate as endoderm and mesoderm to increase relative to the number that differentiate as ectoderm. cDNA probes show that treatment with TPA causes an increase in accumulation of RNAs specific to endoderm and mesoderm with a concomitant decrease in RNAs specific to ectoderm. Treatment of isolated prospective ectodermal cells with TPA causes them to differentiate into endoderm and mesoderm. The critical period for TPA to alter development is during early to mid cleavage, and treatment of embryos with TPA after that time has little effect. These results indicate that PKC may play a key role in determining the fate of cells during sea urchin development.     

Midbody sealing after cytokinesis in embryos of the sea urchin Arabacia punctulata

Summary Cytokinesis consists of a contractile phase followed by sealing of the connecting midbody to form two separated cells. To determine how soon the midbody sealed after cleavage furrow contraction, the fluorescent dye Lucifer Yellow CH(457.3 M.W.) was microinjected into cells at various intervals after cleavage had begun. Mitotic PtK₂ cells were recorded with video-microscopy so that daughter cells in the epithelial sheet could be identified for several hours after cell division. One daughter cell of each pair followed was microinjected to determine whether the dye diffused into the other daughter cell. For intervals up to four hours after the beginning of cytokinesis, diffusion took place between daughter cells. After this time the dye did not spread between daughter cells. In sea urchin blastomeres of the first, second and third divisions, Lucifer Yellow passed between daughter blastomeres only during the first 15 min after cytokinesis. If one cell of a two-cell, four-cell or eight-cell embryo was microinjected more than 15 min after the last cleavage, the dye remained in the injected cell and was distributed to all progeny of that cell, resulting in blastulae that were either one-half, one-quarter or one-eighth fluorescent, respectively. Thus, although cleavage furrow contraction takes approximately the same amount of time in sea urchin blastomeres and PtK₂ cells, the time of midbody sealing differs dramatically in the two cell types. Our results also indicate the importance of knowing the mitotic history of cells when injecting dyes into interphase cells for the purpose of detecting gap junctions.     

Lineage-specific alteration in cell cycle structure in early Tubifex embryos

Embryos of the freshwater oligochaete Tubifex exhibit asynchrony in division timing as early as the second cleavage; this cleavage asynchrony becomes pronounced as development proceeds. The present study was undertaken to elucidate the composition and duration of the cell cycles of early Tubifex embryos, with special reference to their cell lineages. No significant variations in lengths of cleavage cycles were found among early embryos. In all blastomeres up to the eighth cleavage cycle, the M phase was followed directly by a 30 min S phase, which suggested that early embryos lack G1 phase. The durations of the M phase did not change during this period of development, but did differ between cell lines. The M phase in the A and B cell lines lasted for about 130 min, while the M phase in the C and D cell lines lasted for about 95 min. An examination of chromosome cycles showed that this difference in M phase durations resulted from a longer stay by the A/B cell lines in prometaphase. Only G2 phase lengthened during early development. After several rounds of G2 phase extension, three classes of G2 phase duration were established: the most extended G2 phase (∼6 h) in the first quartette of micromeres (cells 1 a–1 d), the shortest G2 phase (∼1.58 h) in teloblasts, and an intermediate G2 phase (∼2.4 h) in the progeny of macromeres (i.e. endodermal cells). Experiments with syncytial blastomeres showed that the timing of entry into the M phase, hence the duration of the G2 phase, was affected by cytoplasmic compositions. The shortest G2 phase correlated closely with the presence of yolk-free cytoplasm called pole plasm.