The movements and fusion of the pronuclei at fertilization of the sea urchin Lytechinus variegatus: Time-lapse video microscopy

The penetration of the sperm into the egg, and the movements of the male and female pronuclei were followed from sperm attachment through pronuclear fusion, using time-lapse video microscopy of gametes and zygotes of the sea urchin Lytechinus variegatus (23° C). The pronuclei move in four stages: I. Sperm Entry Phase, following sperm-egg fusion and a rapid radiating surface contraction (5.9 ± 1.3 μm/second) when egg microvilli engulf the sperm head, midpiece, and tail to form the fertilization cone and the sperm tail beats in the egg cytoplasm; II. Formation of the Sperm Aster, which pushes the male pronucleus centripetally at a rate of 4.9 ± 1.7 μm/minute starting 4.4 ± 0.5 minutes after sperm-egg fusion, as the male pronucleus undergoes chromatin decondensation; III. Movement of the Female Pronucleus, the greatest and fastest of the pronuclear motions at a rate of 14.6 ± 3.5 μm/minute at 6.8 ± 1.2 minute after sperm-egg fusion, which establishes the contact between the pronuclei; and IV. Centration of the Pronuclei to the egg center at a rate of 2.6 ± 0.9 μm/minute by 14.1 ± 2.6 minutes after sperm-egg fusion. Pronuclear fusion typically occurs after stage IV and proceeds rapidly starting 14.7 ± 3.6 minutes after sperm-egg fusion with the male pronucleus coalescing into the female pronucleus at a rate of 14.2 ± 2.6 μm/minute.     

Sperm incorporation,the pronuclear migrations,and their relation to the establishment of the first embryonic axis: Time-lapse video microscopy of the movements during fertilization of the sea urchin Lytechinus variegatus

The movements during fertilization have been investigated with differential interference optics and recorded by time-lapse video microscopy of the clear egg of the sea urchin Lytechinus variegatus. Sperm-egg binding occurs rapidly, and following a time when the sperm gyrates on the egg surface, gamete fusion occurs. A rapid cortical contraction radiates from the fusion site and is succeeded by the elevation of the fertilization coat. Sperm incorporation occurs in two stages: the fertilization cone enlarges around and above the erect and immotile sperm and then the sperm head, midpiece, and tail are displaced along the subsurface region of the egg at an average rate of 3.5 μm/min. The formation of the sperm aster moves the male pronucleus from the subsurface region of the egg toward the egg center at a rate of 4.9 μm/min. When the rays of the radial sperm aster appear to contact the female pronucleus, the female pronucleus migrates at a rate of 14.6 μm/min to the center of the sperm aster. The now adjacent pronuclei are moved to the egg center by the continuing enlargement of the sperm aster at a rate of 2.6 μm/min. Syngamy is usually preceded by the disassembly of the sperm aster. The centripetal migration of the pronuclei appears involved in the establishment of the first embryonic axis; cleavage occurs within 8° of the direction of this centering motion.     

Intracellular sodium activity in the sea urchin egg during fertilization

Fertilization of the sea urchin egg triggers a sequence of events that are necessary for metabolic derepression and stimulation of proliferation. Changes in intracellular Ca2+ and H+ activities regulate the sequence of events. Intracellular sodium activity is important in the regulation of the intracellular activities of these ions and may directly regulate metabolic events. Using Na+-sensitive microelectrodes we continuously measured the intracellular Na+ activity during fertilization. The results show an increase in intracellular sodium activity medicated by two pathways of Na+ entry: Na+ permeability increase during the fertilization potential and initiation of Na+-H+ exchange activity. Intracellular Na+ activity returned to unfertilized levels by 20 min after fertilization. This decrease was inhibited by ouabain, which suggests the activation of Na+, K+ ATPase during fertilization.     

Cytochalasin B blocks sperm incorporation but allows activation of the sea urchin egg

Cytochalasin B (CB) (2 × 10⁻⁶ M) prevents the incorporation of sperm into the eggs of Lytechinus pictus and Strongylocentrotus purpuratus as judged by light and transmission electron microscopy (TEM). At lower concentrations of CB (2 × 10⁻⁷ M), sperm are successfully incorporated into the egg, but their migration in the area of the egg cortex is impaired. The site of action of CB on the sperm may be on the initial rotation of the sperm nucleus in the cortex; the subsequent migration is not affected by CB. Although sperm incorporation is prevented at the higher CB concentrations, the eggs become activated—as judged by cortical reaction, increased protein synthesis and increased respiration. These findings raise the concept that egg activation by sperm could result from some pre-fusion event and hence that sperm-egg fusion would not be a prerequisite for the triggering of development. An alternative hypothesis is that fusion occurs between the acrosome process membrane and egg membrane, but since CB has destroyed the integrity of the cortex actin, the fusion bridge is so weak that it cannot be maintained without some contractile or cytoskeletal support by the cortex. The sperm may activate the CB-treated egg in the same manner as pricking with a microelectrode sometimes does.     

Sperm velocity and longevity trade off each other and influence fertilization in the sea urchin Lytechinus variegatus

The theoretical prediction that fast sperm should be more effective at fertilizing eggs has never been documented empirically. Interspecific comparisons suggest an inverse relationship between sperm velocity and sperm longevity but this trade-off has never been demonstrated within a species. Here I investigate how sperm velocity and sperm longevity influence the patterns of fertilization in the sea urchin Lytechinus variegatus. In the laboratory I examined 11 male female pairs of sea urchins for variation in sperm velocity and sperm longevity, and determined the correlations of these traits with the percentage of eggs fertilized with serially diluted sperm. Males with faster sperm had higher rates of fertilization than males with slower sperm. Within individual males, as sperm aged they slowed down and showed a reduced percentage activity and lower rates of fertilization. Across males, the average velocity of freshly spawned sperm was inversely related to sperm longevity. These results establish the possibility that sperm traits are adapted for varying conditions along a continuum from sperm limitation to sperm competition.     

Integration of sperm and egg plasma membrane components at fertilization

Studies examining the integration of the sperm and egg plasma membranes, subsequent to gamete fusion in the surf clam, Spisula solidissima, were carried out employing the concanavalin A-horseradish peroxidase-diaminobenzidine procedure (Con A-HRP-DAB). When unfertilized Spisula eggs were incubated in Con A, either prior to or after aldehyde fixation and reacted with HRP-DAB, enzymatic precipitate was found associated with the vitelline layer and plasmalemma. The plasma membranes of sperm treated in a similar manner failed to stain. The plasma membranes of fertilized eggs reacted with Con A-HRP-DAB and examined by 1 min postinsemination were associated uniformly with enzymatic precipitate except at sites of sperm incorporation. These portions of unstained plasma membrane were derived from the spermatozoon and delimited the contents of the fertilization cone. From 2 to 4 min postinsemination, HRP-DAB reaction product became associated with the plasma membrane delimiting the fertilization cone. By 4 min postinsemination no difference in staining of the plasma membranes derived from the egg or the sperm (plasmalemma delimiting the fertilization cone) was detected. Evidence is presented suggesting that the acquisition of HRP-DAB reaction product by the former sperm plasmalemma is due to the movement of Con A binding sites from the egg plasma membrane.     

Transmission electron microscopy characterization of macromolecular domain cavities and microstructure of single-crystal calcite tooth plates of the sea urchin Lytechinus variegatus

The calcite plates and prisms in Lytechinus variegatus teeth form a complex biocomposite and employ a myriad of strengthening and toughening strategies. These crystal elements have macromolecule-containing internal cavities that may act to prevent cleavage. Transmission electron microscopy employing a small objective aperture was used to quantify several characteristics of these cavities. Cavity diameters ranged from 10 to 225 nm, the mean cavity diameter was between 50 and 60 nm, and cavities comprised approximately 20% of the volume of the crystal. Some cavities exhibited faceting and trace analysis identified these planes as being predominately of {1014} type. Through focus series of micrographs show the cavities were homogeneously distributed throughout the foil. The electron beam decomposed a substance within cavities and this suggests that these cavities are filled with a hydrated organic phase.     

The distribution of polymerized actin in the rat egg and its sensitivity to cytochalasin B during fertilization

The distribution of polymerized actin in rat eggs fertilized in vitro was determined using NBD-phallacidin (NBD-ph). Unfertilized and fertilized eggs exhibited a 3-5-micron-thick band of fluorescence that encompassed the entire cortical cytoplasm. There was no dramatic increase in the staining of the cortex in association with any component of the fertilizing sperm during its incorporation into the egg. Unfertilized eggs and fertilized eggs obtained at intervals after sperm-egg fusion were treated with cytochalasin B (CB; 5 micrograms/ml) and subsequently stained with NBD-ph. Unfertilized eggs treated with CB exhibited a continuous ring of cortical staining identical to that seen in untreated eggs. Eggs treated with CB 15 min after sperm-egg fusion exhibited small gaps in the cortical staining pattern, whereas those exposed to CB 1 hr after fusion exhibited larger gaps and the staining pattern appeared punctate. This pattern could be seen throughout the remainder of the 7 hr period of sperm incorporation and for at least 13 hr thereafter. CB-treated fertilized eggs that were washed to remove the drug again exhibited uninterrupted cortical staining on treatment with NBD-ph. CB also induced the resorption of surface elevations that are normally seen on the eggs during sperm incorporation, but it did not affect the morphology of unfertilized eggs. The sensitivity to CB during fertilization coincides with the onset of a variety of egg shape changes that occur during the period of sperm incorporation (Battaglia and Gaddum-Rosse, Gamete Res., 10:107-118, 1984a).(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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.     

Surface changes at fertilization: integration of sea urchin (Arbacia punctulata) sperm and oocyte plasma membranes

To examine the integration and fate of the sperm plasma membrane following its incorporation into the oocyte plasma membrane, we have fertilized sea urchin (Arbacia punctulata) gametes reciprocally labeled with cationized ferritin. When unlabeled oocytes were inseminated with labeled sperm, cationized ferritin acceptors moved laterally from the sperm plasma membrane into the fertilization cone and surrounding microvilli, mixing with components of the oocyte plasmalemma. Labeled oocytes inseminated with unlabeled sperm produced extremely large fertilization cones, completely devoid of cationized ferritin, while the remainder of the oocyte surface remained heavily labeled. Surface area measurements indicated that if all the sperm plasmalemma were utilized to delimit a fertilization cone it would provide less than 10% of the total surface membrane. Evidence is presented indicating that a principal source of membrane to the expanding fertilization cone of inseminated oocytes is from microvilli, i.e., microvilli are retracted to accommodate fertilization cone formation. Membrane delimiting the fertilization cone has a much lower affinity for agents (cationized ferritin and concanavalin A) that stain negatively charged and carbohydrate moieties compared to other regions of the oocyte surface. These ultrastructural observations indicate that significant rearrangements occur in the oocyte and sperm plasma membranes following gamete fusion which give rise to asymmetries in membrane topography; components of both membranes are redistributed within the bilayer adjacent to and delimiting the fertilization cone.     

Mechanical properties of the surface of the sea urchin egg at fertilization and during cleavage

1. 1. Changes in stiffness of the cell surface at fertilization and during cleavage in sea urchin eggs were determined by the magnetic particle method.

2. 2. The stiffness of the cell surface increased at fertilization, reached a maximum after about 1.5 min, then decreased and reached a minimum about 4 min after insemination, followed by a gradual increase, in the eggs of Temnopleurus toreumaticus at 25.5 to 26.5 °C.

3. 3. The stiffness of the cell surface increased during the diaster stage, reached a maximum 2 to 3 min before the onset of cleavage, then decreased to a minimum about 1 min before the onset of cleavage, increased again, reached a maximum during cleavage and then diminished, in the eggs of Temnopleurus toreumaticus at 25.5 to 26.5 °C. A similar stiffness change was observed in the eggs of Hemicentrotus pulcherrimus at 17 to 19 °C, occurring almost in parallel in both the equatorial and polar surfaces.

     

K+ activity and regulation of intracellular pH in the sea urchin egg during fertilization

Fertilization of the sea urchin egg is accompanied by changes in intracellular ion activities and transmembrane fluxes, which regulate the sequence of biochemical events of metabolic derepression. Changes in intracellular K+ activity during fertilization have been controversial and here we report our measurements using intracellular K+-sensitive microelectrodes. A small, but statistically significant, transient rise in internal K+ activity was detected during the first 10 min of fertilization. Since this change in K+ activity was ouabain sensitive, intracellular K+ activity in the fertilized egg appears to be regulated by the increased Na+, K+ ATPase activity, rather than the previously suggested K+ decompartmentalization. Increasing external K+ concentration was found to stimulate ouabain-sensitive alkalinization in the fertilized egg. The data are consistent with the possibility that Na+, K+ ATPase may regulate cytoplasmic pH by recycling Na+ that enters the cell through Na+-H+ antiport.     

Radioiodination and characterization of the plasma membrane of sea urchin sperm

Two methods were used to radioiodinate sea urchin sperm: lactoperoxidase-glucose oxidase and Iodo-Gen. Following iodination the sperm are viable, they undergo the acrosome reaction, and they fertilize eggs. Of the radioactivity associated with the labeled sperm, 28–50% is presumed to be free ¹²⁵I^?, 37–47% is incorporated in lipid, and 8–15% is in trypsin-digestible material believed to be protein. Digestion of the labeled, living sperm with trypsin removes 95.6–99.5% of the macromolecular label (the cells are alive after digestion) suggesting that almost all the protein label is on the external surface of the cell. Thin-layer chromatography of the lipid fraction shows that the major membrane phospholipids and cholesterol are labeled. SDS-PAGE analysis shows the protein-incorporated ¹²⁵I is distributed among four glycoproteins of >250K, 84K, 64K, and 52K dalton apparent molecular weight. Twenty-eight percent of the total protein (trypsin-digestible) label is in the 84K component and 46% in the 64K band. Although both molecules contain much of the label, they are relatively minor components of the TX-100 extract of sperm. The methods outlined will be useful in determining the role of sperm surface components in fertilization.     

Bioelectric responses at fertilization: Separation of the events associated with insemination from those due to the cortical reaction in sea urchin,Lytechinus variegatus

The bioelectric responses at fertilization of the sea urchin Lytechinus variegatus are a complex series of membrane potential and resistance changes that occur concomitant with gamete fusion, ionic fluxes, and the cortical granule discharge. This work attempts to separate the electrical effects of sperm-egg interactions from those of the cortical reactions. Two approaches were taken to discern the electrical events associated with insemination, distinct from cortical granule discharge: (1) fertilization of eggs treated with 3% urethane, 10 mM procaine, or 10 mM nicotine, to prevent the cortical reaction and (2) refertilization of fertilized eggs (denuded with 1 mM aminotriazole containing 1 mg/ml soybean trypsin inhibitor). Cortical granule discharge in the absence of sperm incorporation was investigated by artificial activation with 5 μM A23187 or by fertilization in the presence of 10 μM cytochalasin D, which prevents incorporation. These results are consistent with a model in which the sperm-egg interaction triggers both a rapid (50–400 msec), but minor (?10 mV), electrical transient that leads to an action potential and then both the Na⁺-dependent fast block to polyspermy and the late block resulting from the secretion of the cortical granules.     

Isolation and partial characterization of the plasma membrane of the sea urchin egg

The cell surface complex (Detering et al., 1977, J. Cell Biol. 75, 899-914) of the sea urchin egg consists of two subcellular organelles: the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles. We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization. Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na+/K+ ATPase, as well as two cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles. The purified plasma membranes are highly enriched in the Na+/K+ ATPase but contain only very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a "fluffy" proteinaceous layer on the external surface, which probably represent peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with Kl removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and Kl-extracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as a large number of minor species, that are not extractable with Kl. The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least six to seven polypeptide components. The phospholipid composition of the Kl-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was found to be a major component of the plasma membrane. Before Kl extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation.     

The effects of organic and inorganic phosphates on fertilization and early development in the sea urchin Lytechinus variegatus (Echinodermata: Echinoidea)

The embryos of the sea urchin Lytechinus variegatus are capable of surviving chronic exposure to inorganic sodium phosphate and organic triethyl phosphate concentrations as high as 6 and 1000 mg l(-1) seawater, respectively. However, chronic exposure to sub-lethal concentrations of these phosphates may cause arrested or abnormal embryonic development. We measured fertilization success and percentages of normal, arrested and abnormal embryos exposed to low, medium and high sub-lethal concentrations of inorganic and organic phosphate. Fertilization success was significantly reduced in all phosphate treatments. After attaining the 4-cell stage, embryos exposed to the highest phosphate concentrations displayed arrested development. Percentages of abnormally developing embryos showed a strong concentration dose-response with a significant increase in abnormal embryonic development with increasing phosphate concentration. Overall, these results indicate that the gametes and embryos of L. variegatus may provide a rapid and sensitive model bioassay for the evaluation of phosphate pollutants in marine systems. Our findings also indicate that shallow-water populations of L. variegatus spawning in areas subjected to inorganic and organic phosphate pollutants may suffer detrimental effects on fertilization and embryonic development.     

Fusion of membranes during fertilization. Increases of the sea urchin egg''s membrane capacitance and membrane conductance at the site of contact with the sperm

The early events of fertilization that precede and cause activation of an egg have not been fully elucidated. The earliest electrophysiological change in the sea urchin egg is a sperm-evoked increase of the egg's membrane conductance. The resulting depolarization facilitates entry of the fertilizing sperm and precludes the entry of supernumerary sperm. The sequence of the increase in the egg's membrane conductance, gamete membrane fusion, egg activation, and sperm entry, including causal relationships between these events, are not known. This study reports the use of whole egg voltage clamp and loose patch clamp to monitor simultaneously changes of membrane conductance and capacitance at the site of sperm-egg contact. Measurements were made during sperm-egg interactions where sperm entry readily proceeded or was precluded by maintaining the egg's membrane potential either at large, negative values or at positive values. Whenever the sperm evoked an increase of the egg's membrane conductance, that increase initiated abruptly, was localized to the site of sperm attachment, and was accompanied by a simultaneous abrupt increase of the membrane capacitance. This increase of capacitance indicated the establishment of electrical continuity between gametes (possibly fusion of the gametes' plasma membranes). If sperm entry was blocked by large negative membrane potentials, the capacitance cut off rapidly and simultaneously with a decrease of the membrane conductance, indicating that electrical continuity between gametes was disrupted. When sperm entry was precluded by positive membrane potentials, neither conductance nor capacitance increased, indicating that sperm entry was halted before the fusion of membranes. A second, smooth increase of capacitance was associated with the exocytosis of cortical granules near the sperm in eggs that were activated. Electrical continuity between the gametes always preceded activation of the egg, but transient electrical continuity between the gametes alone was not always sufficient to induce activation.     

Ion channel activity of membrane vesicles released from sea urchin sperm during the acrosome reaction

The sperm acrosome reaction (AR) involves ion channel activation. In sea urchin sperm, the AR requires Ca2+ and Na+ influx and K+ and H+ efflux. During the AR, the plasma membrane fuses with the acrosomal vesicle membrane forming hybrid membrane vesicles that are released from sperm into the medium. This paper reports the isolation and preliminary characterization of these acrosome reaction vesicles (ARVs), using synaptosome-associated protein of 25 kDa (SNAP-25) as a marker. Isolated ARVs have a unique protein composition. The exocytosis regulatory proteins vesicle-associated membrane protein and SNAP-25 are inside ARVs, as judged by protease protection experiments, and membrane associated based on Triton X-114 partitioning. ARVs fused with planar bilayers display three main types of single channel activity. The most frequently recorded channel is cationic, weakly voltage dependent and has a low open probability that increases with negative potentials. This channel is activated by cAMP, blocked by Ba2+, and has a PK+/PNa+ selectivity of 4.5. ARVs represent a novel membrane preparation suitable to deepen our understanding of ion channel activity in the AR and during fertilization.     

Isolation and characterization of a plasma membrane fraction from sea urchin sperm exhibiting species specific recognition of the egg surface

A method is described for isolating preparative quantities of plasma membranes from sea urchin sperm. The final membrane fraction is homogeneous by sucrose density sedimentation and is enriched in adenylate cyclase as well as in the four glycoproteins accessible to radioiodination of intact sperm. The electrophoretic profiles of sperm membranes from three sea urchin species are very similar. The membrane preparation consists primarily of sealed vesicles which release carboxyfluorescein when exposed to detergents or distilled water. Ninety-two percent of the 125I-labeled vesicle material binds to wheat germ lectin columns, suggesting a right-side-out orientation. The isolated sperm membrane vesicles exhibit species specific adhesion to the surfaces of sea urchin eggs; this adhesion is blocked by pretreatment of the vesicles with trypsin or egg jelly. This method will be useful for isolating biologically active sperm membrane components involved in sperm-egg recognition during fertilization.