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.     

Calcium influx following fertilization of Urechis caupo eggs.

Measurements of ⁴⁵Ca flux into and out of Urechis eggs indicate that, during the first 10 min after insemination, the eggs take up 0.24 pmole of Ca/egg. Total egg Ca measured by atomic absorption (AA) spectroscopy increased by 0.23 pmole of Ca/egg (0.56, 0.79, and 0.76 pmole of Ca/egg for unfertilized, 10-min fertilized, and 60-min fertilized eggs, respectively). Thus, the total change in egg Ca is accounted for by the influx even though the rate of efflux, measured as a release of ⁴⁵Ca from preloaded eggs, increases to twice the unfertilized rate by 15 min. The fertilization influx follows saturation kinetics (K_a = 1.3 mM). It is competitively inhibited by procaine, but is not inhibited by dinitrophenol, mersalyl acid, or ruthenium red. Ten percent of the total Ca influx has occurred by 10 sec, and it is, therefore, the most rapid response to fertilization yet known in these eggs. The influx is also observed in eggs partially activated by insemination in pH 7 seawater (SW); the other fertilization responses, except sperm penetration, do not occur in pH 7 SW. Although Ca influx alone is insufficient to activate the eggs, it may be a prerequisite for cytoplasmic activation and development, inducing other secondary responses which are prevented by low external pH.     

Repair of a genetically-caused defect in oogenesis in Drosophila melanogaster by transplantation of cytoplasm from wild-type eggs and by injection of pyrimidine nucleosides

Eggs produced by homozygous mutant rudimentary (r⁹, 154.5) females of Drosophila melanogaster die during embryogenesis, apparently because the mutant female fails to incorporate certain substances into the egg during oogenesis. These eggs can be rescued by injecting them at the preblastoderm stage with wild-type nuclei and cytoplasm or wild-type cytoplasm alone from unfertilized eggs. Some preblastoderm eggs injected with 1.5% of egg volume of cytoplasm from unfertilized wild-type eggs were able to complete both embryonic and postembryonic development and emerged as adults, whereas not a single uninjected control egg was able to complete embryonic development. The eggs of rudimentary mothers can also be rescued by injecting each egg at the blastoderm stage with 0.01 μg of pyrimidine nucleosides. The results demonstrate that a pyrimidine deficiency is the cause of abortive embryogenesis, and confirm the previous finding that the rudimentary mutation leads to pyrimidine auxotrophy.     

Thymidine kinase activation in unfertilized sea urchin eggs by homogenization and fertilization

Kinetics of in vivo phosphorylation of ³H-thymidine taken up by sea urchin eggs was compared between unfertilized and fertilized eggs. The percentage of phosphorylated ³H-thymidine in the total acid-soluble radioactivity in the cell increased with increasing incubation time within the first several minutes of incubation in the unfertilized eggs, while nearly 100% of phosphorylation of thymidine was observed without regards to the incubation time and in spite of a tremendous increase in the net uptake of thymidine in the fertilized eggs, suggesting possible activation of thymidine kinase occurring soon after fertilization.In contrast to the in vivo finding, the thymidine kinase activity in unfertilized egg homogenates was found in general to be almost as large as that in fertilized egg homogenates. However, when the enzyme activity was assayed within a short period (30 min) after homogenization of unfertilized eggs, the activity was found to increase more or less with time after homogenization, reaching a level equal to that in fertilized egg homogenates. This enzyme activation after homogenization was especially marked in case of Pseudocentrotus eggs and sometimes amounted to a several fold increase.Preliminary investigations revealed possible involvement of some redox reaction(s) in the thymidine kinase activation during and/or after homogenization of unfertilized sea urchin eggs.     

The cytolytic isolation of the cortex of the sea urchin egg

When the vitelline layer of sea urchin eggs (Lytechinus pictus) is disrupted by trypsin or dithiothreitol and the eggs are placed in an isosmotic medium devoid of Ca²⁺, cytolysis of the eggs occurs. During lysis the entire egg cortex peels off in one piece. Lysis is temperature and pH dependent and is inhibited by cytochalasin B. Cortices from unfertilized eggs contain seven major macromolecular components. A 42K-dalton component is believed to be actin, representing between 12 and 27% of the total protein. Cortices from fertilized eggs may contain between 50 and 65% actin. The actin appears to increase the strength of its attachment to the cortex after fertilization. This method of isolating the entire cortex may be useful for studying structural and enzymatic changes which may occur in the cortex during the cell cycle.     

Changes of uridine permeability during the maturation of tunicate eggs.

The time course of uridine uptake by eggs and embryos of the tunicate Ascidia callosa was studied using 5-min pulses of [³H]uridine at intervals from the unfertilized egg to the 16-cell embryo. The unfertilized egg is permeable to uridine, but 5 min after fertilization uptake begins to drop, reaching a minimum of 30% of the unfertilized rate about 30 min after fertilization. At 45 min after fertilization, permeability begins to increase, reaching a plateau about 3 hr after fertilization at the two-cell stage. The initial decrease in permeability occurs at first polar body production; the increase at 45 min is coincident with the formation of the second polar body. Substrate concentration experiments up to 200 μM show strict concentration dependence for uridine uptake. The inhibitors p-chloromercuribenzoate (PCMB), dinitrophenol (DNP), and thymidine have little, if any effect on permeability. Cold (?1°C) and Na⁺-free sea water inhibit uptake 60% during all three developmental stages. The changes in permeability may be indicative of temporary reorganization of the plasma membrane during the fertilization-initiated completion of meiosis.     

Polyspermic fertilization of sea urchin eggs treated with protease inhibitors: Localization of sperm receptor sites at the egg surface

The normal elevation of the fertilization membrane and the establishment of the block to polyspermy are retarded in Arbacia punctulata eggs by specific protease inhibitors, soybean trypsin inhibitor (SBTI), leupeptin, and antipain. Ultrastructural observations show that the vitelline layer remains attached to the plasma membrane of fertilized SBTI treated eggs at numerous sites (cortical projections). Quantitive morphometric analysis indicates that the vitelline layer elevates from about 65% of the surface of SBTI treated eggs during the first 3 min post insemination. However, the vulnerability of SBTI treated eggs to refertilization (polyspermy) only declined during the subsequent gradual detachment of the vitelline layer from the cortical projections over the next 15 min. Antipain and leupeptin (10^?5 to 10^?3M) also promoted polyspermy in Arbacia eggs by a process of refertilization extending for a 10- to 15-min period after the initial monospermic insemination. Normal cleavage and development was obtained when eggs were placed in leupeptin and antipain (10^?3M) after the fertilization membrane had elevated. The data indicate that the normal secretory function (or functions) of the cortical granule protease in establishing the block to polyspermy is retarded by these protease inhibitors, and that the vitelline layer is transformed into a mechanical barrier to prevent penetration by supernumerary sperm during its detachment from the plasma membrane of the egg. Furthermore, the vitelline layer in unfertilized eggs appears to be a mosaic structure, with sperm receptor sites localized in regions of the egg's surface, which give rise to cortical projections in the presence of SBTI.     

Compartmentation of thymidine kinase in unfertilized sea urchin eggs and possible release of thymidine kinase from particulates in activated eggs

The thymidine kinase activity of homogenates of unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, in 1 M NaCl was always lower than that of homogenates of the unfertilized eggs in hypotonic media or homogenates of the fertilized or ammonia-activated eggs in 1 M NaCl by 30–50%. Sonication of the unfertilized egg homogenates in 1 M NaCl resulted in the elevation of thymidine kinase activity up to a level in the fertilized or ammonia-activated egg homogenates which is not affected by sonication. Differential centrifugation of unfertilized egg homogenates in 1 M NaCl revealed that the latent thymidine kinase is associated with the 1500g pellet or even with the 200g pellet. Exposure of the 1500g pellet to sonication, hypotonic media, 0.3% Triton X-100 in 1 M NaCl, and 2 M propyleneglycol resulted in the elevation of thymidine kinase, which was eventually shown to be no longer bound to the pellet fraction. Latent thymidine kinase was not detected in the 1500g pellet prepared from the fertilized egg homogenate in 1 M NaCl. These findings seem to suggest that thymidine kinase in unfertilized eggs may be sequestered, at least partly, in some large intracellular structures but may be released from them upon fertilization or ammonia activation, in accordance with our earlier observation on the apparent activation of thymidine kinase afer fertilization.     

Properties of adenylate cyclase activity during early sea urchin development

The total adenylate cyclase activity in homogenates of eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus was assayed in vitro and found to remain constant in eggs before and at intervals after fertilization. In S. purpuratus egg homogenates virtually all of the enzyme activity was sedimented by centrifugation at 20 000 g. The enzyme specific activity in the 20 000 g pellet remained unchanged at each point through first cleavage, though it was several-fold higher than in the whole homogenate. The adenylate cyclase from both fertilized and unfertilized eggs was maximally active in vitro when assayed with 10 mM MgSO₄ and 10 mM NaF at pH 8 using 0.2 mM AMP-PNP (an ATP analog) as the substrate. Sucrose density gradient centrifugation of egg homogenates showed that adenylate cyclase activity was present in fractions which sedimented at a variety of densities. The adenylate cyclase specific activity in cortices isolated by the method of Sakai [10] from eggs at first cleavage was 4- to 6-fold higher than in unfertilized egg cortices. The increased enzyme activity in egg cortices at first cleavage suggests that adenylate cyclase-containing membranes may become localized within the egg cortex after fertilization.     

Filipin/sterol complexes in fertilized and unfertilized sea urchin egg membranes

Sea urchin (Arbacia punctulata) eggs and zygotes were treated with filipin in an effort to examine changes in membrane sterols at fertilization. The plasma membrane of treated unfertilized eggs possessed numerous filipin/sterol complexes, while fewer complexes were associated with membranes delimiting cortical granules, demonstrating that the plasmalemma is relatively rich in β-hydroxysterols in comparison to cortical granule membrane. Following fusion with the plasmalemma, membrane formerly delimiting cortical granules underwent a dramatic alteration in sterol composition, as indicated by a rapid increase in the number of filipin/sterol complexes. In contrast, portions of the zygote plasma membrane, derived from the plasmalemma of the unfertilized egg, displayed little or no change in filipin/sterol composition. Other than regions of the plasma membrane engaged in endocytosis, the plasmalemma of the zygote possessed a homogeneous distribution of filipin/sterol complexes and appeared similar to that of the unfertilized egg. These results demonstrate that following its fusion with the egg plasmalemma, membranes, formerly delimiting cortical granules, undergo a dramatic alteration in sterol composition. Changes in the localization of filipin/sterol complexes are discussed in reference to alterations in egg plasmalemmal function at fertilization.     

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.     

Fertilization-induced changes in concanavalin A binding to mouse eggs

The binding of concanavalin A (ConA) to zona-free unfertilized and fertilized mouse eggs has been investigated using tritiated ConA. At low lectin concentrations (1–5 μg ml^?1) the fertilized egg shows a higher affinity for [³H]ConA than does the unfertilized egg. In saturation conditions, however, unfertilized and fertilized eggs show the same binding capacity (1.55 × 10⁸ ConA molecules/egg). The results indicate that ConA-binding sites change qualitatively following fertilization; possible connections between this change and other fertilization-induced changes in the egg surface are discussed.     

Lectins induce the redistribution and internalization of receptors on the surface of cultured neurons

A cytoplasmic dynein ATPase has been identified in three species of unfertilized sea urchin eggs, Strongylocentrotus droebachiensis, S. purpuratus, and Arbacia punctulata. The enzyme was partially purified by sucrose gradient density centrifugation, and its polypeptide chain weight and composition were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein has enzymatic characteristics in common with flagellar dynein. It is activated nearly equally by Mg²⁺ and Ca²⁺, has no activity in the presence of K⁺ and EDTA, shows a specificity for ATP over other nucleoside triphosphates, and is inhibited by vanadate anion. On SDS-PAGE, the enzyme shows two major bands at 320,000 and 385,000 daltons, comigrating with certain ciliary and flagellar dynein polypeptides. The enzyme, given the name “egg dynein,” constitutes 2 to 4% of the total cell protein in the unfertilized egg and maintains this high value from fertilization through the late blastula stage. It appears to be equally distributed throughout the embryo at the 16-cell stage. Possible functions of egg dynein are discussed and models for dynein-microtubule mediated movements within the cytoplasm are presented.     

Assessing the In Situ Fertilization Status of Two Marine Copepod Species,Temora longicornis and Eurytemora herdmani; How Common Are Unfertilized Eggs in Nature?

We utilized an egg staining technique to measure the in situ fertilization success of two marine copepod species, Temora longicornis and Eurytemora herdmani from May to October 2008 in coastal Maine and correlated fertilization success with environmental conditions in their habitat. T. longicornis is a free spawning species that releases eggs into the ambient seawater after mating. In contrast, E. herdmani carries eggs in an egg sac until they hatch. The proportion of fertilized eggs within E. herdmani egg sacs was significantly higher than the freely spawned clutches of T. longicornis. This may be a result of the asymmetrical costs associated with carrying vs. spawning unfertilized eggs. T. longicornis frequently laid both fertilized and unfertilized eggs within their clutch. T. longicornis fertilization was negatively associated with chlorophyll concentration and positively associated with population density in their local habitat. The fertilization status of E. herdmani egg sacs was high throughout the season, but the proportion of ovigerous females was negatively associated with an interaction between predators and the proportion of females in the population. This study emphasizes that, in addition to population level processes, community and ecosystem level processes strongly influence the fertilization success and subsequent productivity of copepods.     

Changes in the organization of the mouse egg plasma membrane upon fertilization and first cleavage: Indications from the lateral diffusion rates of fluorescent lipid analogs

Using the technique of fluorescence photobleaching recovery, we have measured the lateral diffusion of the lipid analogs 3,3′-diacylindocarbocyanine iodides of acyl chain lengths, n = 14 to 22 in the plasma membranes of unfertilized and fertilized CD-1 mouse eggs and two-cell embryos. Mean diffusion coefficients ranged from (3.4 to 7.9) × 10^?9 cm²/sec and mean percentage diffusing ranged from 65 to 82%. Within 10% diffusing fractions are independent of chain length and unaffected by fertilization or first cleavage. In unfertilized and fertilized eggs the diffusion coefficients show relative maxima at n = 16 and 20. In two-cell embryos the relative maximum at n = 16 is suppressed. As was observed by D. E. Wolf, W. Kinsey, W. Lennarz, and M. Edidin (1981, Develop. Biol.81, 133) on sea urchin eggs, the effect of fertilization on diffusion coefficients is chain length dependent. Little change is observed for n = 14 and 18; a slight increase is observed for n = 16; and small decreases are observed for n = 20 and 22. This is interpreted in terms of a reordering of the lipid domains in the plasma membrane upon fertilization and first cleavage.     

Cortical granules remaining after fertilization in Xenopus laevis

Eggs of Xenopus laevis were examined in an electron microscope at unfertilized egg, 1-cell, 2-cell, 32-cell, and blastula stages. Granules closely resembling cortical granules were observed within the “germinal plasm” as well as in the peripheral cytoplasm of all the eggs examined. A staining method was developed that makes it easier to count cortical granules in thick Epon sections. Light and electron microscope examinations revealed that granules remaining after fertilization possessed morphological characteristics wholly consistent with those of cortical granules of unfertilized eggs. These granules were confirmed to be true cortical granules.     

Osmotrophy in fossil protoctists and early animals

Summary

The role of Ca²⁺ in activation and early development of locust eggs was examined through measurement of ooplasmic Ca²⁺ levels before and after fertilization, and through experimental activation of unfertilized eggs. Ooplasmic pCa (i.e. the negative logarithm of Ca²⁺ activity) measured in intact eggs decreased from 5.35 before fertilization, to 4.77 and 3.00 by 1 day and 3 days after fertilization, respectively. pCa was also determined for samples of ooplasm collected by rupturing eggs under paraffin oil. The pCa was 5.10 in ooplasm isolated from unfertilized eggs, and 3.84 in ooplasm collected from eggs within 4 h of fertilization. Ooplasmic pCa remained between 3.97 and 3.12 from 1–6 days after fertilization. Since a decline in pCa indicates an increase in ooplasmic Ca²⁺ activity, the data suggest that regulation of ooplasmic Ca²⁺ during post-fertilization development involves release of Ca²⁺ from internal stores. Experimental egg activation was examined in eggs dissected from the oviducts before fertilization and incubated on moist filter paper. Some eggs were first immersed in experimental solutions for 30–60 minutes before incubation. The presence of an embryo 2 or 4 days after fertilization or experimental treatment was used as an indicator of egg activation. Activation occurred in 92% and 12% of fertilized and untreated eggs, respectively. The percentage of unfertilized eggs which activated increased to 47% if eggs were soaked 30–60 minutes in physiological saline, and to as much as 65%-68% if eggs were injected with Ca²⁺ buffers or if a Ca²⁺ action potential was evoked. Up to 36% and 42% of unfertilized eggs activated after incubation in Ca²⁺-free salines or in the presence of the Ca²⁺-channel blocker Cd²⁺, respectively. Taken together, the results suggest that entry of external Ca²⁺ through voltage dependent channels increases the proportion of eggs which activate, but is not an absolute requirement for activation.     

Regeneration of sialic acid on the surface of Chinese hamster cells in culture. I. General characteristics of the replacement process

Electropotential differences between the cell interior and the external medium have been studied with intracellular microelectrodes in ovarian oocytes, ovulated unfertilized eggs and fertilized eggs of R. pipiens. In ovarian oocytes the cytoplasm was 50 to 80 mV negative, relative to isotonic Ringer's solution. In contrast, electrode penetration of the oocyte nucleus in situ indicated that the nucleoplasm was about 25 mV positive, relative to the cytoplasm. After ovulation, the cortical cytoplasm became 20 to 50 mV positive with regard to an external solution of 0.1 strength Ringer's solution (ca. pond water). Penetration of the cytoplasm at levels from 0.3 to 0.6 mm below the egg surface revealed an inner zone with a potential which was about 15 mV negative, relative to the cortical cytoplasm. A slow hyperpolarization of the cortical membrane occurred at activation, with the potential returning to that of the ovulated unfertilized egg within ten minutes. After fertilization, the egg cytoplasm remained positive until the first cleavage. As division proceeded, the cytoplasm slowly depolarized and became 50 to 60 mV negative, relative to 0.1 strength Ringer's solution.     

The ontogeny of germ plasm during oogenesis in Drosophila

Primordial germ cells can be induced at both the anterior and ventral region of the Drosophila egg by transplanted posterior polar plasm. Two questions arise from these results: (1) Is fertilization required for germ plasm to be functional, and (2) at what stage during oogenesis does the posterior polar plasm become established as a germ-cell determinant?Polar plasm from unfertilized eggs and from oocytes at stage 10 to 14 of Drosophila melanogaster was implanted into the anterior region of cleavage embryos. Some injected embryos were analyzed at the ultrastructural level during blastoderm formation. Polar plasm from unfertilized eggs and from oocytes of stages 13 and 14 was found to be integrated into several anterior cells that resembled morphologically normal pole cells. The formation of such cells, however, could not be detected in embryos injected with polar plasm from oogenetic stages 10 to 12. Experimentally induced pole cells proved to be capable of differentiating into functional germ cells when cycled through the germ line of genetically different host embryos. About 5% of the flies developing from these embryos produced progeny that originated from the induced pole cells. Germ-line mosaicism in those flies also could be detected histochemically in their gonads. No germ cells were recovered with polar plasm transplants from oogenetic stages 10 to 12.The results show that posterior polar plasm of the unfertilized egg is functional in germ-cell determination, and that prior to egg maturation this cytoplasm has already acquired its determinative ability. This is the first demonstration that specific developmental information stored in the cytoplasm can be traced back to a particular region of the oocyte.     

A trypsin-like proteinase localized in cortical granules isolated from unfertilized sea urchin eggs by zonal centrifugation. Role of the enzyme in fertilization

A trypsin-like proteinase was localized within a single subcellular compartment of unfertilized Strongylocentrotus purpuratus eggs, the cortical granules. Homogenates of eggs were fractionated by rate-zonal centrifugation. Enzymatic markers were used to determine the distribution of mitochondria (cytochrome oxidase), yolk platelets (acid nitrophenyl phosphatase), and cortical granules (β-1, 3-glucanase) in the sucrose density gradient. A bimodal distribution pattern was obtained for aryl esterase activity (substrate: β-naphthyl acetate), with one peak in the microsomal and the other in the cortical granule fractions. The cortical granule enzyme was characterized as a trypsin-like proteinase, since it also hydrolyzed another typical tryptic substrate α-N-benzoyl-l-arginine ethyl ester and was completely inactivated by soybean trypsin inhibitor (SBTI). The aryl esterase activity in the microsomal fractions was not inhibited by SBTI, while 50% of the total aryl esterase activity in the original egg homogenate was inactivated by SBTI. The identity of the enzyme(s) responsible for the aryl esterase activity associated with the microsomal particles is unknown at present.The cortical granule proteinase functions in the elevation of the fertilization membrane and establishment of the block to polyspermy at fertilization. Arbacia punctulata eggs inseminated in the presence of trypsin inhibitors, SBTI or tosyl lysine chloromethyl ketone (TLCK), failed to elevate normal fertilization membranes and became heavily polyspermic.On the basis of these results and observations made by other investigators with a wide variety of biological systems, it is proposed that trypsin-like proteinases function in the discharge of secretory granules from all types of cells.