scholarly journals Nuclear envelope breakdown is under nuclear not cytoplasmic control in sea urchin zygotes.

1995 ◽  
Vol 129 (6) ◽  
pp. 1447-1458 ◽  
Author(s):  
G Sluder ◽  
E A Thompson ◽  
C L Rieder ◽  
F J Miller
Keyword(s):  
Nuclear Envelope ◽  
Dna Synthesis ◽  
Sea Urchin ◽  
Control Mechanisms ◽  
Checkpoint Control ◽  
Intact Male ◽  
Male Pronucleus ◽  
Nuclear Envelope Breakdown ◽  
Sperm Nuclei ◽  
Histone Kinase

Nuclear envelope breakdown (NEB) and entry into mitosis are though to be driven by the activation of the p34cdc2-cyclin B kinase complex or mitosis promoting factor (MPF). Checkpoint control mechanisms that monitor essential preparatory events for mitosis, such as DNA replication, are thought to prevent entry into mitosis by downregulating MPF activation until these events are completed. Thus, we were surprised to find that when pronuclear fusion in sea urchin zygotes is blocked with Colcemid, the female pronucleus consistently breaks down before the male pronucleus. This is not due to regional differences in the time of MPF activation, because pronuclei touching each other break down asynchronously to the same extent. To test whether NEB is controlled at the nuclear or cytoplasmic level, we activated the checkpoint for the completion of DNA synthesis separately in female and male pronuclei by treating either eggs or sperm before fertilization with psoralen to covalently cross-link base-paired strands of DNA. When only the maternal DNA is cross-linked, the male pronucleus breaks down first. When the sperm DNA is cross-linked, male pronuclear breakdown is substantially delayed relative to female pronuclear breakdown and sometimes does not occur. Inactivation of the Colcemid after female NEB in such zygotes with touching pronuclei yields a functional spindle composed of maternal chromosomes and paternal centrosomes. The intact male pronucleus remains located at one aster throughout mitosis. In other experiments, when psoralen-treated sperm nuclei, over 90% of the zygote nuclei do not break down for at least 2 h after the controls even though H1 histone kinase activity gradually rises close to, or higher than, control mitotic levels. The same is true for normal zygotes treated with aphidicolin to block DNA synthesis. From these results, we conclude that NEB in sea urchin zygotes is controlled at the nuclear, not cytoplasmic, level, and that mitotic levels of cytoplasmic MPF activity are not sufficient to drive NEB for a nucleus that is under checkpoint control. Our results also demonstrate that the checkpoint for the completion of DNA synthesis inhibits NEB by acting primarily within the nucleus, not by downregulating the activity of cytoplasmic MPF.

scholarly journals Nucleo-cytoplasmic interactions that control nuclear envelope breakdown and entry into mitosis in the sea urchin zygote

1999 ◽  
Vol 112 (8) ◽  
pp. 1139-1148 ◽  
Author(s):  
E.H. Hinchcliffe ◽  
E.A. Thompson ◽  
F.J. Miller ◽  
J. Yang ◽  
G. Sluder
Keyword(s):  
Nuclear Envelope ◽  
Dna Synthesis ◽  
Sea Urchin ◽  
Mammalian Cells ◽  
Cyclin B1 ◽  
Nuclear Accumulation ◽  
Dna Breaks ◽  
Male Pronucleus ◽  
Nuclear Envelope Breakdown ◽  
Female Pronucleus

In sea urchin zygotes and mammalian cells nuclear envelope breakdown (NEB) is not driven simply by a rise in cytoplasmic cyclin dependent kinase 1-cyclin B (Cdk1-B) activity; the checkpoint monitoring DNA synthesis can prevent NEB in the face of mitotic levels of Cdk1-B. Using sea urchin zygotes we investigated whether this checkpoint prevents NEB by restricting import of regulatory proteins into the nucleus. We find that cyclin B1-GFP accumulates in nuclei that cannot complete DNA synthesis and do not break down. Thus, this checkpoint limits NEB downstream of both the cytoplasmic activation and nuclear accumulation of Cdk1-B1. In separate experiments we fertilize sea urchin eggs with sperm whose DNA has been covalently cross-linked to inhibit replication. When the pronuclei fuse, the resulting zygote nucleus does not break down for >180 minutes (equivalent to three cell cycles), even though Cdk1-B activity rises to greater than mitotic levels. If pronuclear fusion is prevented, then the female pronucleus breaks down at the normal time (average 68 minutes) and the male pronucleus with cross-linked DNA breaks down 16 minutes later. This male pronucleus has a functional checkpoint because it does not break down for >120 minutes if the female pronucleus is removed just prior to NEB. These results reveal the existence of an activity released by the female pronucleus upon its breakdown, that overrides the checkpoint in the male pronucleus and induces NEB. Microinjecting wheat germ agglutinin into binucleate zygotes reveals that this activity involves molecules that must be actively translocated into the male pronucleus.

scholarly journals An ultrastructural study of cross-fertilization (Arbacia female x Mytilus male).

1977 ◽  
Vol 73 (1) ◽  
pp. 14-26 ◽  
Author(s):  
F J Longo
Keyword(s):  
Sea Urchin ◽  
Ultrastructural Study ◽  
Cortical Granule ◽  
Male Pronucleus ◽  
Female Pronucleus ◽  
Sperm Nuclei ◽  
Cross Fertilization ◽  
Sperm Aster

Insemination of sea urchin (Arbacia) ova with mussel (Mytilus) sperm has been accomplished by treating eggs with trypsin and suspending the gametes in seawater made alkaline with NaOH. Not all inseminated eggs undergo a cortical granule reaction. Some eggs either elevate what remains of their vitelline layer or demonstrate no cortical modification whatsoever. After its incorporation into the egg, the nucleus of Mytilus sperm undergoes changes which eventually give rise to the formation of a male pronucleus. Concomitant with these transformations, a sperm aster may develop in association with the centrioles brought into the egg with the spermatozoon. Both the male pronucleus and the sperm aster may then migrate centrad to the female pronucleus. Evidence is presented which suggests that fusion of the male pronuclei from Mytilus sperm with female pronuclei from Arbacia eggs may occur, although this was not directly observed. These results demonstrate that Mytilus sperm nuclei are able to react to conditions within Arbacia eggs and differentiate into male pronuclei.

scholarly journals Role of spindle microtubules in the control of cell cycle timing.

1979 ◽  
Vol 80 (3) ◽  
pp. 674-691 ◽  
Author(s):  
G Sluder
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Control Cell ◽  
Microtubule Assembly ◽  
Nuclear Envelope Breakdown ◽  
Anaphase Onset ◽  
Spindle Cells ◽  
Spindle Microtubules

Sea urchin eggs are used to investigate the involvement of spindle microtubules in the mechanisms that control the timing of cell cycle events. Eggs are treated for 4 min with Colcemid at prophase of the first mitosis. No microtubules are assembled for at least 3 h, and the eggs do not divide. These eggs show repeated cycles of nuclear envelope breakdown (NEB) and nuclear envelope reformation (NER). Mitosis (NEB to NER) is twice as long in Colcemid-treated eggs as in the untreated controls. Interphase (NER to NEB) is the same in both. Thus, each cycle is prolonged entirely in mitosis. The chromosomes of treated eggs condense and eventually split into separate chromatids which do not move apart. This "canaphase" splitting is substantially delayed relative to anaphase onset in the control eggs. Treated eggs are irradiated after NEB with 366-nm light to inactivate the Colcemid. This allows the eggs to assemble normal spindles and divide. Up to 14 min after NEB, delays in the start of microtubule assembly give equal delays in anaphase onset, cleavage, and the events of the following cell cycle. Regardless of the delay, anaphase follows irradiation by the normal prometaphase duration. The quantity of spindle microtubules also influences the timing of mitotic events. Short Colcemid treatments administered in prophase of second division cause eggs to assemble small spindles. One blastomere is irradiated after NEB to provide a control cell with a normal-sized spindle. Cells with diminished spindles always initiate anaphase later than their controls. Telophase events are correspondingly delayed. This work demonstrates that spindle microtubules are involved in the mechanisms that control the time when the cell will initiate anaphase, finish mitosis, and start the next cell cycle.

scholarly journals Experimental manipulation of the amount of tubulin available for assembly into the spindle of dividing sea urchin eggs.

1976 ◽  
Vol 70 (1) ◽  
pp. 75-85 ◽  
Author(s):  
G Sluder
Keyword(s):  
Nuclear Envelope ◽  
Sea Urchin ◽  
Maximum Rate ◽  
Spindle Assembly ◽  
Experimental Manipulation ◽  
Lytechinus Variegatus ◽  
Tubulin Polymerization ◽  
Treated Cell ◽  
Sea Urchin Eggs ◽  
Nuclear Envelope Breakdown

Spindle assembly is studied in the eggs of the sea urchin Lytechinus variegatus by experimentally varying the amount of polymerizable tubulin within the egg. Aliquots of fertilized eggs from the same female are individually pulsed for 1-6 min with 1 X 10(-6) M Colcemid at least 20 min before first nuclear envelope breakdown. This treatment inactivates a portion of the cellular tubulin before the spindle is formed. Upon entering mitosis, treated eggs form functional spindles that are reduced in length and birefringent retardation but not width. With increased exposure to Colcemid, the length and retardation of the metaphase spindles are progressively reduced. Similar results are obtained by pulsing the eggs with Colcemid before fertilization, which demonstrates that the tubulin found in unfertilized sea urchin eggs is later used in spindle formation. Spindles, once assembled, are responsive to increases in the amount of polymerizable tubulin within the cell. Rapid increases in the amount of polymerizable tubulin within a Colcemid-treated cell can be experimentally effected by irradiating the cells with 366-nm light. This treatment photochemically inactivates the Colcemid, thereby freeing the tubulin to polymerize. Upon irradiation, the small prometaphase spindles of Colcemid-treated cells immediately increase in length and retardation. In these irradiated cells, spindle length and retardation increase as much as four times faster than they do during prometaphase for normal spindles. This suggests that the rate of the normal prometaphase increase in retardation and spindle size may be determined by factors other than the maximum rate of tubulin polymerization in the cell.

scholarly journals Dynamics of the Endoplasmic Reticulum and Golgi Apparatus during Early Sea Urchin Development

2000 ◽  
Vol 11 (3) ◽  
pp. 897-914 ◽  
Author(s):  
Mark Terasaki
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Permeability Barrier ◽  
Cell Cycles ◽  
Nuclear Envelope Breakdown ◽  
Quantitative Measurements ◽  
Green Fluorescent

The endoplasmic reticulum (ER) and Golgi were labeled by green fluorescent protein chimeras and observed by time-lapse confocal microscopy during the rapid cell cycles of sea urchin embryos. The ER undergoes a cyclical microtubule-dependent accumulation at the mitotic poles and by photobleaching experiments remains continuous through the cell cycle. Finger-like indentations of the nuclear envelope near the mitotic poles appear 2–3 min before the permeability barrier of the nuclear envelope begins to change. This permeability change in turn is ∼30 s before nuclear envelope breakdown. During interphase, there are many scattered, disconnected Golgi stacks throughout the cytoplasm, which appear as 1- to 2-μm fluorescent spots. The number of Golgi spots begins to decline soon after nuclear envelope breakdown, reaches a minimum soon after cytokinesis, and then rapidly increases. At higher magnification, smaller spots are seen, along with increased fluorescence in the ER. Quantitative measurements, along with nocodazole and photobleaching experiments, are consistent with a redistribution of some of the Golgi to the ER during mitosis. The scattered Golgi coalesce into a single large aggregate during the interphase after the ninth embryonic cleavage; this is likely to be preparatory for secretion of the hatching enzyme during the following cleavage cycle.

Intracellular free calcium rise triggers nuclear envelope breakdown in the sea urchin embryo

Nature ◽  
1988 ◽  
Vol 332 (6162) ◽  
pp. 364-366 ◽  
Author(s):  
Richard A. Steinhardt ◽  
Janet Alderton
Keyword(s):  
Nuclear Envelope ◽  
Sea Urchin ◽  
Intracellular Free Calcium ◽  
Free Calcium ◽  
Nuclear Envelope Breakdown ◽  
Sea Urchin Embryo

Decondensation of the mouse sperm nucleus within the interphase nucleus

Zygote ◽  
1998 ◽  
Vol 6 (1) ◽  
pp. 39-45 ◽  
Author(s):  
Yasutaka Maeda ◽  
Hiroko Yanagimachi ◽  
Hiroyuki Tateno ◽  
Noriko Usui ◽  
R. Yanagimachi
Keyword(s):  
Nuclear Envelope ◽  
Interphase Nucleus ◽  
Sperm Nucleus ◽  
Interphase Nuclei ◽  
Mouse Sperm ◽  
Immature Oocytes ◽  
Nuclear Envelope Breakdown ◽  
Germinal Vesicles ◽  
Sperm Nuclei ◽  
Mouse Eggs

SummarySperm nuclei incorporated into the cytoplasm (ooplasm) of fertilised mouse eggs at the pronuclear stage remain condensed, whereas those injected into male or female pronuclei decondense. Similarly, sperm nuclei injected into germinal vesicles of immature oocytes or the nuclei of 2-cell embryos decondense, while those entering the cytoplasm of these oocytes / embryos do not. These facts seem to suggest that factors necessary for the decondensation of sperm nucleus are present in interphase nuclei and are released into the ooplasm during nuclear envelope breakdown. Nucleoplasmin, which is synthesised in the cytoplasm and accumulated within the nucleus, is likely a major candidate for these factors.

scholarly journals Multifunctional Ca2+/calmodulin-dependent protein kinase is necessary for nuclear envelope breakdown.

1990 ◽  
Vol 111 (5) ◽  
pp. 1763-1773 ◽  
Author(s):  
C Baitinger ◽  
J Alderton ◽  
M Poenie ◽  
H Schulman ◽  
R A Steinhardt
Keyword(s):  
Protein Kinase ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Mitotic Division ◽  
Cam Kinase ◽  
Dependent Protein Kinase ◽  
Sea Urchin Eggs ◽  
Nuclear Envelope Breakdown ◽  
Dependent Protein

The role of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in nuclear envelope breakdown (NEB) was investigated in sea urchin eggs. The eggs contain a 56-kD polypeptide which appears to be a homologue of neuronal CaM kinase. For example, it undergoes Ca2+/calmodulin-dependent autophosphorylation that converts it to a Ca2(+)-independent species, a hallmark of multifunctional CaM kinase. It is homologous to the alpha subunit of rat brain CaM kinase. Autophosphorylation and substrate phosphorylation by the sea urchin egg kinase are inhibited in vitro by CaMK(273-302), a synthetic peptide corresponding to the autoinhibitory domain of the neuronal CaM kinase. This peptide inhibited NEB when microinjected into sea urchin eggs. Only one mAb to the neuronal enzyme immunoprecipitated the 56-kD polypeptide. Only this antibody blocked or significantly delayed NEB when microinjected into sea urchin eggs. These results suggest that sea urchin eggs contain multifunctional CaM kinase, and that this enzyme is involved in the control of NEB during mitotic division.

Lithium blocks cell cycle transitions in the first cell cycles of sea urchin embryos, an effect rescued by myo-inositol

Development ◽  
1997 ◽  
Vol 124 (6) ◽  
pp. 1099-1107 ◽  
Author(s):  
A. Becchetti ◽  
M. Whitaker
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Sea Water ◽  
Cleavage Stage ◽  
Teratogenic Effects ◽  
Cell Cycles ◽  
Lytechinus Pictus ◽  
Nuclear Envelope Breakdown ◽  
Phosphoinositide Pathway

Lithium is a classical inhibitor of the phosphoinositide pathway and is teratogenic. We report the effects of lithium on the first cell cycles of sea urchin (Lytechinus pictus) embryos. Embryos cultured in 400 mM lithium chloride sea water showed marked delay to the cell cycle and a tendency to arrest prior to nuclear envelope breakdown, at metaphase and at cytokinesis. After removal of lithium, the block was reversed and embryos developed to form normal late blastulae. The lithium-induced block was also reversed by myo- but not epi-inositol, indicating that lithium was acting via the phosphoinositide pathway. Lithium microinjection before fertilization caused arrest prior to nuclear envelope breakdown at much lower concentrations (3-5 mM). Co-injection of myo-inositol prevented the block. Microinjection of 1–2 mM lithium led to block at the cleavage stage. This was also reversed by coinjection of myo-inositol. Embryos blocked by lithium microinjection proceeded rapidly into mitosis after photolysis of caged inositol 1,4,5-trisphosphate. These data demonstrate that a patent phosphoinositide signalling pathway is essential for the proper timing of cell cycle transitions and offer a possible explanation for lithium's teratogenic effects.

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