Characterization of water in unfertilized and fertilized sea urchin eggs

The water in unfertilized and fertilized sea urchin eggs was characterized with a proton nuclear magnetic resonance (NMR) titration method assuming fast proton diffusion (FPD) between water compartments. This method involves stepwise dehydration with sequential T1 relaxation time and water content determinations. The results analyzed by the FPD model give evidence of intracellular water compartments with three different correlation times: 6 X 10(-12) sec (bulk water), 1 X 10(-10) sec (structured water) and about 2 X 10(-9) sec (bound water). Fertilization is accompanied by a substantial increase in bulk water (from 111 to 414 g H2O per 100 g dry mass) and by a decrease in the water of hydration (from 128 g to 56 g per 100 g dry mass). This study shows that 54% of the water in the unfertilized sea urchin egg has motional properties different from bulk water and that this percentage decreases dramatically shortly after fertilization. Most of the change in T1 relaxation rate observed at fertilization can be accounted for by uptake of bulk water associated with elevation of the fertilization membrane.     

Surface area change at fertilization: Resorption of the mosaic membrane

Eggs of Strongylocentrotus purpuratus (sea urchin) have a surface area of 41,000 μm² before fertilization as determined by quantitative transmission and scanning electron microscopy. Within a minute after fertilization 18,000 cortical vesicles contribute an additional 57,000 μm² to form a mosaic membrane with the original plasma membrane. However, by 16 min after fertilization the total area of the egg is only 45,000 μm², indicating a rapid resorption of surface. Calculations of surface area depend in large part upon the numbers and dimensions of microvilli, after careful compensations are made for specimen shrinkage. The 134,000 microvilli per egg are 0.35 μm long before fertilization. They elongate to 1.0 μm in the first few minutes and then soon shorten to 0.5 μm. Even at their longest, microvilli do not accommodate all of the surface area of cortical vesicle membrane. The merger of cortical vesicle membranes and the plasma membrane was demonstrated many years ago and is not in doubt; however, this study indicates that the resulting mosaic membrane is not a long-lived, simple arithmetic combination of its components. Rather, the mosaic membrane undergoes a rapid and dynamic shrinkage by a mechanism which is not apparent on the basis of egg topography alone. The absolute values of egg surface area and dynamic changes in the surface are discussed in relation to physiological events accompanying fertilization.     

CHANGES OF THE MEMBRANE POTENTIAL AT THE TIME OF FERTILIZATION IN THE SEA URCHIN EGG WITH SPECIAL REFERENCE TO THE FERTILIZATION-WAVE

An experimental device was developed from the work of U ehara and S ugiyama (1969), in order to study the electrical phenomena accompanying the fertilization-wave in the sea urchin egg.
The change in membrane potential upon fertilization consists of 2 peaks (I to et al. , 1970), being preceded by a shoulder. The shoulder appears within the "latent period" (A llen and G riffin , 1958), and the 2 peaks correspond to the breakdown of the cortical granules and the formation of the fertilization membrane.
When the equatorial region of the egg surface was exposed to a detergent-sea water, the breakdown of the cortical granules and the formation of the fertilization membrane are induced only in this ring-shaped area. Sperm is then added to one of the polar regions. The fertilization-wave, starting from the point of sperm-entry, propagates across the detergent-treated region, and the membrane is formed on the whole egg surface. During such an experiment, changes of the membrane potential in the detergent-treated region were measured. 1 to 3 sudden transient depolarizations appear, followed by a delayed small depolarization. It is presumed that the initial depolarization corresponds to the fertilization-wave. The pattern of the potential change at normal fertilization may be explained by complexity of the cortical change, and the initial depolarizing shoulder is considered to correspond to the fertilization-wave, which is isolated by the above-mentioned device.     

Mechanism for Increase in Intracellular Concentration of Free Calcium in Fertilized Sea Urchin Egg : A Method for Estimating Intracellular Concentration of Free Calcium

Intracellular free calcium concentration in the sea urchin egg was calculated to increase from 0.1 mM in an unfertilized egg to 1 mM in a fertilized egg 10 min after fertilization, based on measurement of the dissociation constant between free calcium and sea urchin egg homogenate. The dissociation constant between free calcium (dialyzable calcium) and homogenate of sea urchin eggs was measured by means of dialysis equilibrium. The dissociation constant of the unfertilized egg was about 10^–4 M and that of the fertilized egg was about 10–3 M in three species of sea urchin, Hemicentrotus pulcherrimus, Anthocidaris crassispina, and Pseudocentrotus depressus. An increase in the dissociation constant of the unfertilized egg homogenate was observed after the addition of calcium ion at a concentration above 0.3 mM, the dissociation constant becoming the same as that observed in the fertilized egg homogenate after the administration of CaCl₂ at a concentration above 1 mM. Sodium ion also caused a decrease in the calcium-binding ability of the unfertilized egg homogenate. Therefore, penetration of calcium ion or sodium ion upon fertilization might induce an increase in the dissociation constant and then intracellular concentration of free calcium would increase at fertilization. Almost all calcium-binding ability of the egg homogenate was found in the microsomal fraction, and the substance which bound calcium was thought to be protein in nature, since trypsin could decrease the level of calcium-binding substance in the homogenate of the eggs.     

Inhibition of sea urchin fertilization by jaspisin, a specific inhibitor of matrix metalloendoproteinase

Jaspisin, originally isolated from a marine sponge as an inhibitor of the hatching of the sea urchin (Hemicentrotus pulcherrimus) embryo, causes inhibition of sea urchin fertilization. Electron microscopic examination revealed that the acrosome reaction was induced in jaspisin-treated sperm when they were incubated with an intact egg. The acrosome-reacted sperm bound to the vitelline layer by the acrosomal material surrounding the acrosomal process. However, fusion of the acrosomal process and the egg plasma membrane failed to take place. Membrane potential changes were monitored using eggs preloaded with a membrane potential-sensitive fluorochrome, di-8-ANEPPS. Depolarization of the membrane potential, normally observed in the fertilized egg was not observed in the egg inseminated in the presence of jaspisin, indicating the absence of electrical continuity between the jaspisin-treated egg and sperm. Jaspisin inhibited the activities of matrix metallo-endoproteinase members but not of other types of proteinases. These results provide strong, albeit indirect, evidence that a matrix metallo-endoproteinase(s) is involved in the process of gamete fusion during sea urchin fertilization.     

STUDIES ON SULFHYDRYL GROUPS DURING CELL DIVISION OF SEA URCHIN EGG : II. Mass Isolation of the Egg Cortex and Change in Its—SH Groups during Cell Division

Masses of cortices of both unfertilized and fertilized sea urchin eggs can be isolated by crushing eggs in hypotonic MaCl₂ (0.1 M) solution. The amount of cortical material in terms of protein-N increases steadily after fertilization until the monaster stage and thereafter remains almost constant until well into the two-cell stage. The amount of bound—SH per protein-N of the egg cortex also increases after fertilization, reaches a maximum value at the amphiaster stage and thereafter decreases rapidly as the cleavage of the cell proceeds.     

Studies of the mechanism of the electrical polyspermy block using voltage clamp during cross-species fertilization

Prevention of polyspermic fertilization in sea urchins (Jaffe, 1976, Nature (Lond.). 261:68-71) and the worm Urechis (Gould-Somero, Jaffe, and Holland, 1979, J. Cell Biol. 82:426-440) involves an electrically mediated fast block. The fertilizing sperm causes a positive shift in the egg's membrane potential; this fertilization potential prevents additional sperm entries. Since in Urechis the egg membrane potential required to prevent fertilization is more positive than in the sea urchin, we tested whether in a cross-species fertilization the blocking voltage is determined by the species of the egg or by the species of the sperm. With some sea urchin (Strongylocentrotus purpuratus) females, greater than or equal to 90% of the eggs were fertilized by Urechis sperm; a fertilization potential occurred, the fertilization envelope elevated, and sometimes decondensing Urechis sperm nuclei were found in the egg cytoplasm. After insemination of sea urchin eggs with Urechis sperm during voltage clamp at +50 mV, fertilization (fertilization envelope elevation) occurred in only nine of twenty trials, whereas, at +20 mV, fertilization occurred in ten of ten trials. With the same concentration of sea urchin sperm, fertilization of sea urchin eggs occurred, in only two of ten trials at +20 mV. These results indicate that the blocking voltage for fertilization in these crosses is determined by the sperm species, consistent with the hypothesis that the fertilization potential may block the translocation within the egg membrane of a positively charged component of the sperm.     

EXPERIMENTAL STUDIES ON THE SITE OF PROPAGATION OF THE FERTILIZATION-WAVE IN THE SEA URCHIN EGG

Although the fertilization-wave in the sea urchin egg is generally considered to propagate over the egg surface, there has been no definite evidence to show the site of propagation. The possibility that the wave passes through the endoplasm, not over the egg surface, has not been denied.
A drop of paraffin was injected into an egg, so that the endoplasm was divided into 2 parts by the paraffin drop, the 2 parts being connected only by the egg cortex. When spermatozoa were added to one side of the egg, the fertilization membrane was formed first on this part of the egg and then on the opposite part. This indicates that the egg surface or the egg cortex is the site of propagation of the fertilization-wave and the endoplasm has no direct influence on the propagation.     

Temporal sequence and spatial distribution of early events of fertilization in single sea urchin eggs

Measurements and observations of five early events of fertilization, singly and in pairs, from single sea urchin eggs have revealed the precise temporal sequence and spatial distribution of these events. In the Arbacia punctulata egg, a wave of surface contraction occurs coincident with membrane depolarization (t = 0). These two earliest events are followed by the onset of a rapid, propagated increase in cytoplasmic-free calcium at approximately 23 s as measured by calcium- aequorin luminescence. The luminescence reaches its peak value by 40 s after the membrane depolarization. The luminescence remains uniformly elevated for some time before its decay over several minutes. The onset of an increase in the pyridine nucleotide (NAD(P)H) fluorescence follows the membrane depolarization at approximately 51 s. The fertilization membrane begins its elevation in a wave-like fashion coincidentally with the increase in NAD(P)H fluorescence. Similar results are observed in the Lytechinus variegatus egg. The results suggest that while the increase in cytoplasmic-free calcium may be important for many changes occurring in the egg, the elevated-free calcium is not directly responsible for the propagated wave of cortical granule exocytosis.     

Cross fertilization between sea urchin eggs and oyster spermatozoa

Interphylum crossing was examined between sea urchin eggs (Temnopleurus hardwicki) and oyster sperm (Crassostrea gigas). The eggs could receive the spermatozoa with or without cortical change. The fertilized eggs that elevated the fertilization envelope began their embryogenesis. Electron microscopy revealed that oyster spermatozoa underwent acrosome reaction on the sea urchin vitelline coat, and their acrosomal membrane fused with the egg plasma membrane after the appearance of an intricate membranous structure in the boundary between the acrosomal process and the egg cytoplasm. Oyster spermatozoa penetrated sometimes into sea urchin eggs without stimulating cortical granule discharge and consequently without fertilization envelope formation. The organelles derived from oyster spermatozoa seemed to be functionally inactive in the eggs whose cortex remained unchanged.     

MORPHOLOGY OF GAMETE MEMBRANE FUSION AND OF SPERM ENTRY INTO OOCYTES OF THE SEA URCHIN

Sea urchin gametes predominate in molecular studies of fertilization, yet relatively little is known of the subcellular aspects of sperm entry in this group. Accordingly, it seemed desirable to make a detailed examination of sperm entry phenomena in sea urchins with the electron microscope. Gametes of the sea urchins Arbacia punctulata and Lytechinus variegatus were used in this study. Samples of eggs containing 2 to 8 per cent oocytes were selected and fixed with osmium tetroxide in sea water at various intervals after insemination. Fixed specimens were embedded in Epon 812, sectioned, and examined with an electron microscope. An apical vesicle was observed at the anterior end of the acrosome. The presence of this structure, together with other observations, suggested that initiation of the acrosome reaction in sea urchin sperm involves dehiscence of the acrosomal region with the subsequent release of the acrosomal granule. Contact and initial fusion of gamete membranes was observed in mature eggs and oocytes and invariably involved the extended acrosomal tubule of the spermatozoon. Only one spermatozoon normally enters the mature egg. The probability of locating such a sperm in ultrathin sections is exceedingly low. Several sperm do normally enter oocytes. Consequently, observations of sperm entry were primarily restricted to the latter. The manner of sperm entry into oocytes did not resemble phagocytosis. Organelles of the spermatozoon were progressively divested of their plasma membrane as they entered the ground cytoplasm of the oocyte fertilization cone. Initiation of the acrosome reaction, contact and initial fusion of gamete membranes, and sperm entry into oocytes of sea urchins conform to the Hydroides-Saccoglossus pattern of early fertilization events as described by Colwin and Colwin (13).     

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.     

Direct membrane retrieval into large vesicles after exocytosis in sea urchin eggs

At fertilization in sea urchin eggs, elevated cytosolic Ca2+ leads to the exocytosis of 15,000-18,000 1.3-microns-diam cortical secretory granules to form the fertilization envelope. Cortical granule exocytosis more than doubles the surface area of the egg. It is thought that much of the added membrane is retrieved by subsequent endocytosis. We have investigated how this is achieved by activating eggs in the presence of aqueous- and lipid-phase fluorescent dyes. We find rapid endocytosis of membrane into 1.5-microns-diam vesicles starting immediately after cortical granule exocytosis and persisting over the following 15 min. The magnitude of this membrane retrieval can compensate for the changes in the plasma membrane of the egg caused by exocytosis. This membrane retrieval is not stimulated by PMA treatment which activates the endocytosis of clathrin-coated vesicles. When eggs are treated with short wave-length ultraviolet light, cortical granule exocytosis still occurs, but granule cores fail to disperse. After egg activation, large vesicles containing semi-intact cortical granule protein cores are observed. These data together with experiments using sequential pulses of fluid-phase markers support the hypothesis that the bulk of membrane retrieval immediately after cortical granule exocytosis is achieved through direct retrieval into large endocytotic structures.     

ELECTRICAL POLYSPERMY BLOCK IN SEA URCHINS: NICOTINE AND LOW SODIUM EXPERIMENTS1

Nicotine reduces the amplitude of the fertilization potential in sea urchin eggs, at least in part because it decreases the slope of the current voltage relation of the unfertilized egg membrane. The reduced fertilization potential amplitude provides an electrophysiological explanation for previous observations that nicotine impairs the fast block to polyspermy. The block to polyspermy is also impaired by fertilization in low sodium sea water, a medium which has been reported to reduce fertilization potential amplitude.     

Microvilli in sea urchin eggs : Differences in their formation and type

The elongation of microvilli, which normally occurs upon fertilization in sea urchin eggs, was also observed in unfertilized eggs treated with the enzyme, papain. Cortical granule exocytosis, which is thought to be the source of membrane used in microvillar elongation, does not occur in the papain-treated eggs. It appears, therefore, that there is more than one way in which the egg plasma membrane can increase very quickly and to a great extent. In addition, the kinds of microvilli formed in the two instances appear to be different. Previous work with reaggregating sea urchin cells is also cited to support the suggestion that microvilli can form in different ways and are of different types.     

INDUCTION OF THE ACROSOME REACTION ON THE EGG SURFACE OF DE-JELLIED SEA URCHIN EGGS BEFORE AND AFTER FERTILIZATION*

The capacity of the surface of sea urchin eggs to induce the acrosome reaction was assayed by estimating the rate of acrosome reaction of supernumerary spermatozoa in the presence of variously treated eggs before and after fertilization. DTT-disruption of the vitelline coat did not eliminate the acrosome reaction-inducing capacity. This capacity was retained after fertilization in eggs of both H. pulcherrimus and A. crassispina. The acrosome reaction-inducing capacity of the eggs was markedly decreased by treatment with trypsin. The low capacity of the trypsin-treated eggs was maintained after fertilization in H. pulcherrimus, but in A. crassispina the capacity returned to the pre-trypsin treatment level after fertilization. Fertilized eggs from which the fertilization membrane was mechanically removed retained the inducing capacity to a considerable extent, independent of the presence or absence of the hyaline layer, but the capacity diminished rapidly as cleavage proceeded. It was concluded from these data that the acrosome reaction of spermatozoa actually occurred at the surface of de-jellied eggs and that the inducing substance resides in the plasma membrane in addition to the fertilization membrane. A chemical difference between the inducing substance of egg surface and jelly substance is discussed.     

An intermediate state of fertilization involved in internalization of sperm components

The pathway of sperm entry during sea urchin fertilization was analyzed by using sperm covalently labeled with fluorescent and radioactive tracers. Sperm that have been covalently labeled on their surfaces with fluorescein isothiocyanate (FITC) or a radioactive congener, diiodofluorescein isothiocyanate (¹²⁵IFC), transfer labeled components to the egg that persist throughout early development. In order to study the transfer of sperm components and their fate after fertilization, cytochalasin B-dependent inhibition of fertilization, previously shown to permit the cortical reaction of sea urchin eggs but block sperm pronuclear incorporation, was investigated. Under certain conditions cytochalasin B or D (CB or CD) results in about half of the activated eggs having both the sperm nucleus and the fluorescently labeled sperm components arrested apparently at the level of the egg plasma membrane. This arrest of internalization was reversed by removal of CB or CD, and the sperm derivatives entered the egg. When sperm were labeled noncovalently with ethidium bromide or rhodamine 123, fluorescence was transferred to the egg in the cytochalasin-inhibited state in a fashion similar to that found in normal fertilization; in both cases the sperm fluorescence disappeared within a few minutes of fertilization, due to the repartitioning of the noncovalent dyes into the egg cytoplasm. It is concluded that cytochalasin arrests fertilization at an intermediate step in which the sperm has fused with the egg to achieve cytoplasmic continuity, but in which the subsequent internalization of sperm components is inhibited. After removal of cytochalasins the fluorescent sperm components move from the egg surface to an internal site, a process that can be monitored by time-lapse video microscopy with an image intensifier to permit extended observations of sperm fluorescence. The cytoplasmic location of labeled sperm components was substantiated by autoradiography of early embryos fertilized with ¹²⁵IFC-labeled sperm; transfer of sperm components to an internal site was seen after fertilization of either sea urchin or mouse eggs. Taken together, the data suggest that the fate of the labeled sperm surface components, as well as that of the sperm nucleus, is to be transferred to the egg cytoplasm, and that this transfer is mediated by the actin-dependent cytoskeleton of the egg.     

Microvilli on sea urchin eggs: a second burst of elongation

A scanning EM study reveals about 300,000 microvilli on each egg of the sea urchin Strongylocentrotus droebachiensis. The microvilli are about 0.2 μm long before fertilization, elongate to about 0.5 μm soon after fertilization (the “first burst” of microvillus elongation), and subsequently elongate again about midway between fertilization and first cell division (the “second burst” of elongation). The second burst occurs during a discrete 30-min period and results in some microvilli being as long as 10 μm, although the average length is about 1.8 μm. The surface area of the egg following the second burst is about 2.7 times the area of the unfertilized egg.     

Proteolytic cleavage of the cell surface protein p160 is required for detachment of the fertilization envelope in the sea urchin

Sea urchin eggs secrete a serine protease activity, CGSP1, at fertilization that is essential for the block to polyspermy. Several targets of this proteolytic activity on the plasma membrane were identified here using a cell surface biotinylation approach. Amino acid microsequencing of one of these proteins led to the identification of a 4.75-kb cDNA clone from a Strongylocentrotus purpuratus ovary cDNA library that encodes a 160-kDa protein called p160. This protein contains five CUB domains and a putative transmembrane domain suggesting that p160 is an integral membrane protein with protein-protein interaction motifs facing the extracellular matrix of the egg. Whole-mount immunolocalization studies demonstrate that p160 is on the surface of the egg, enriched at the tips of microvilli. The protein is removed at fertilization in a protease-dependent manner, and functional assays suggest that p160 serves to link the plasma membrane to the vitelline layer until fertilization. Thus, p160 is a key candidate for a vitelline-layer linker protein, the selective proteolysis of which functions in the block to polyspermy in the sea urchin egg.     

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.