scholarly journals THE FINE STRUCTURE OF PRONUCLEAR DEVELOPMENT AND FUSION IN THE SEA URCHIN, ARBACIA PUNCTULATA

1968 ◽  
Vol 39 (2) ◽  
pp. 339-368 ◽  
Author(s):  
Frank J. Longo ◽  
Everett Anderson
Keyword(s):  
Nuclear Envelope ◽  
Sea Urchin ◽  
Plasma Membranes ◽  
Light And Electron Microscopy ◽  
Sperm Nucleus ◽  
Sperm Chromatin ◽  
Zygote Nucleus ◽  
Arbacia Punctulata ◽  
Male Pronucleus ◽  
Sperm Aster

Fertilization events following coalescence of the gamete plasma membranes and culminating in the formation of the zygote nucleus were investigated by light and electron microscopy in the sea urchin, Arbacia punctulata. Shortly after the spermatozoon passes through the fertilization cone, it rotates approximately 180° and comes to rest lateral to its point of entrance. Concomitantly, the nonperforated nuclear envelope of the sperm nucleus undergoes degeneration followed by dispersal of the sperm chromatin and development of the pronuclear envelope. During this reorganization of the sperm nucleus, the sperm aster is formed. The latter is composed of ooplasmic lamellar structures and fasciles of microtubules. The male pronucleus, sperm mitochondrion, and flagellum accompany the sperm aster during its migration. As the pronuclei encounter one another, the surface of the female pronucleus proximal to the advancing male pronucleus becomes highly convoluted. Subsequently, the formation of the zygote nucleus commences with the fusion of the outer and the inner membranes of the pronuclear envelopes, thereby producing a small internuclear bridge and one continuous, perforated zygote nuclear envelope.

scholarly journals Organization of the sea urchin egg endoplasmic reticulum and its reorganization at fertilization.

1991 ◽  
Vol 114 (5) ◽  
pp. 929-940 ◽  
Author(s):  
M Terasaki ◽  
L A Jaffe
Keyword(s):  
Sea Urchin ◽  
Time Lapse ◽  
Sperm Nucleus ◽  
Temporal Organization ◽  
Calcium Wave ◽  
Zygote Nucleus ◽  
Lytechinus Pictus ◽  
Sea Urchin Egg ◽  
A Cell ◽  
Sperm Aster

The ER of eggs of the sea urchin Lytechinus pictus was stained by microinjecting a saturated solution of the fluorescent dicarbocyanine DiIC18(3) (DiI) in soybean oil; the dye spread from the oil drop into ER membranes throughout the egg but not into other organelles. Confocal microscopy revealed large cisternae extending throughout the interior of the egg and a tubular membrane network at the cortex. Since diffusion of DiI is confined to continuous bilayers, the spread of the dye supports the concept that the ER is a cell-wide, interconnected compartment. In time lapse observations, the internal cisternae were seen to be in continuous motion, while the cortical ER was stationary. After fertilization, the internal ER appeared to become more finely divided, beginning as a wave apparently coincident with the calcium wave and becoming most marked by 2-3 min. By 5-8 min the ER returned to an organization similar to that of the unfertilized egg. The cortical network also changed at fertilization; it became disrupted and eventually recovered. DiI labeling allowed continuous observations of the ER during pronuclear migration and mitosis. DiI-stained membranes accumulated in the region of the microtubule array surrounding the sperm nucleus and centriole (the sperm aster) as it migrated to the center of the egg; this accumulation persisted near the centrosomes and zygote nucleus throughout pronuclear fusion and the first two mitotic cycles. We have used a new method to observe the spatial and temporal organization of the ER in a living cell, and we have demonstrated a striking reorganization of the ER at fertilization.

scholarly journals THE EFFECTS OF NICOTINE ON FERTILIZATION IN THE SEA URCHIN, ARBACIA PUNCTULATA

1970 ◽  
Vol 46 (2) ◽  
pp. 308-325 ◽  
Author(s):  
Frank J. Longo ◽  
Everett Anderson
Keyword(s):  
Sea Urchin ◽  
Cortical Response ◽  
Large Nucleus ◽  
Zygote Nucleus ◽  
Arbacia Punctulata ◽  
Male Pronucleus ◽  
Initial Series ◽  
Female Pronucleus

The number of sperm incorporated into eggs made polyspermic with varying concentrations of nicotine (0.025–0.25%, v/v) appears to be directly related to the concentrations employed. The cortical response is morphologically equivalent to that observed in control preparations. Shortly after their incorporation all of the spermatozoa undergo structural events normally associated with the development of the male pronucleus in monospermic eggs. During the reorganization of the spermatozoa, sperm asters are formed. The number of male pronuclei that initially migrate to and encounter the female pronucleus is usually one to three. When pronuclei come into proximity to one another the surface of the female pronucleus proximal to the advancing male pronuclei flattens and becomes highly convoluted. Subsequently, the pronuclei contact each other and the outer and inner membranes of the pronuclear envelopes fuse, thereby producing the zygote nucleus. The male pronuclei remaining in the zygote after this initial series of pronuclear fusions continue to differentiate, i.e. they enlarge, form nucleolus-like bodies, and undergo further chromatin dispersion. In approximately 90% of the zygotes, all of the remaining male pronuclei progressively migrate to the zygote nucleus and fuse to form one large nucleus by 80 min postinsemination. Mitosis and cleavage of the polyspermic zygote occurs later than in monospermic eggs.

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.

Sperm nuclear envelope: breakdown of intrinsic envelope and de novo formation in hamster oocytes or eggs

Zygote ◽  
1997 ◽  
Vol 5 (1) ◽  
pp. 35-46 ◽  
Author(s):  
Noriko Usui ◽  
Atsuo Ogura ◽  
Yasuyuki Kimura ◽  
Ryuzo Yanagimachi
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
De Novo ◽  
Germinal Vesicle ◽  
Mature Oocyte ◽  
Sperm Nucleus ◽  
Sperm Chromatin ◽  
Early Prophase ◽  
Nuclear Envelope Breakdown ◽  
M Phase

SummaryDuring fertilisation of a fully mature oocyte, the sperm intrinsic nuclear envelope (SINE) disappears soon after sperm-oocyte fusion. A new nuclear envelope appears around the decondensed sperm chromatin when the oocyte reaches telophase II. Whether the SINE persists or rapidly disappears after sperm entery into immature oocytes or fertilised eggs has been controversial. Nuclear envelopes have been demonstrated around the sperm chromatin, which cannot be decondensed within the ooplasm of these oocytes or eggs, but whether these envelopes are persisting SINEs or newly formed envelopes has been apoint of dispute. To resolve this issue, the fate of the germinal vesicle stage(GV oocytes) or fertilised eggs at the pronuclear stage(PN eggs). The SINEs disappeared quikly within these oocytes or eggs, like those within maturing or mature oocytes, suggesting that the envelops around the sperm chromatin must be newly formed after SINE breakdown. To obtain further evidence, a detergent-treated, SINE-free sperm nucleus was injected into a PN egg. A new envelope appeared around the still-condensed or partially decondensed sperm chromatin within 3h after injection. Thus, disassembly of the SINE within ooplasm, unlike that of nuclear envelopes of other cells at prophase, is independent of the cell cycle stage of the oocyte or egg, whereas the ability of the ooplasm to assemble the new envelope is restricted to certain periods of the cycle. i.e. early prophase and telophase during meiosis and interphase, periods when active M-phase Promoting factor (MPF) is absent from the ooplasm.

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 Targeting of membranes to sea urchin sperm chromatin is mediated by a lamin B receptor-like integral membrane protein.

1996 ◽  
Vol 135 (6) ◽  
pp. 1715-1725 ◽  
Author(s):  
P Collas ◽  
J C Courvalin ◽  
D Poccia
Keyword(s):  
Membrane Protein ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Binding Capacity ◽  
Integral Membrane Protein ◽  
Sperm Chromatin ◽  
Lamin B ◽  
Lamin B Receptor ◽  
Pronuclear Formation

We have identified an integral membrane protein of sea urchin gametes with an apparent molecular mass of 56 kD that cross-reacts with an antibody against the nucleoplasmic NH2-terminal domain of human lamin B receptor (LBR). In mature sperm, p56 is located at the tip and base of the nucleus from where it is removed by egg cytosol in vitro. In the egg, p56 is present in a subset of cytoplasmic membranes (MV2 beta) which contributes the bulk of the nuclear envelope during male pronuclear formation. p56-containing vesicles are required for nuclear envelope assembly and have a chromatin-binding capacity that is mediated by p56. Lamin B is not present in these vesicles and is imported into the nucleus from a soluble pool at a later stage of pronuclear formation. Lamin B incorporation and addition of new membranes are necessary for pronuclear swelling and nuclear envelope growth. We suggest that p56 is a sea urchin LBR homologue that targets membranes to chromatin and later anchors the membrane to the lamina.

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 The Effect of Partial Protein Extraction on the Structure of the Eggs of the Sea Urchin, Arbacia punctulata

1960 ◽  
Vol 7 (1) ◽  
pp. 21-26 ◽  
Author(s):  
R. E. Kane
Keyword(s):  
Nuclear Envelope ◽  
Sea Urchin ◽  
Protein Extraction ◽  
Extraction Procedure ◽  
Cellular Protein ◽  
Soluble Proteins ◽  
Annulate Lamellae ◽  
Cytoplasmic Matrix ◽  
Arbacia Punctulata ◽  
Cell Structures

The structure of the eggs of the sea urchin, Arbacia punctulata, has been investigated after the removal of one-half of the cellular protein. The procedure involves treatment of the eggs with 30 per cent ethanol at -10°C. followed by extraction of the soluble proteins with water. The eggs remain intact, although all of the cytoplasmic matrix is removed. Most cell structures can still be identified, although only the membranes of most remain. The mitochondria lose all of their matrix but retain the inner membranes or cristae. The annulate lamellae appear unaffected by this extraction procedure, remaining intact and apparently undamaged. The nuclear envelope is also retained, although it often undergoes a curious disorganization, apparently as the result of the separation of its two layers. The significance of these observations with respect to the structure of the envelope is discussed.

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