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.     

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.     

An electrical block is required to prevent polyspermy in eggs fertilized by natural mating of Xenopus laevis

In 27% DeBoer's saline (DBS), which yields maximum fertility rates, Xenopus eggs fertilized in vitro are monospermic, regardless of sperm concentration. One block to polyspermy (the “slow” block), described previously, occurs at the fertilization envelope that is elevated in response to the cortical reaction. This paper describes properties of an earlier, “fast” block at the plasma membrane and evaluates the functional significance of the two blocks at physiological sperm concentrations in natural mating conditions. Unfertilized eggs have a resting membrane potential of ?19 mV in 27% DBS. Fertilization triggers a rapid depolarization to +8 mV (the fertilization potential, FP); the potential remains positive for ca. 15 min. Activation of eggs with the ionophore, A23187, produces a slower but similar depolarization (the activation potential, AP). As in other amphibian eggs, the FP appears to result from a net efflux of Cl^?, since the peak of the FP (or the AP in ionophore-activated eggs) decreases as the concentration of chloride salts in the medium is increased. In 67% DBS no FP or AP is observed; eggs fertilized in 67% DBS become polyspermic and average 2 sperm entry sites per egg. In the 5–37 mM range, I^? and Br^?, but not F^?, are more effective than Cl^? in producing polyspermy. In 20 mM NaI the plasma membrane hyperpolarizes in response to sperm or ionophore; 100% levels of polyspermy and an average of 14 sperm entry sites per egg are observed. NaI does not inhibit or retard elevation of the fertilization envelope; the cortical reaction and fertilization envelope are normal in transmission electron micrographs. In 67% DBS, which also inhibits the fast block, the slow block was estimated to become functional 6–8 min after insemination. Eggs fertilized by natural mating in 20 mM NaI exhibit polyspermy levels of 50–90% and average 5 sperm entry sites per egg. Since eggs become polyspermic when fertilized by natural mating under conditions that inhibit the fast, but not the slow, block to polyspermy, we conclude that the fast block is essential to the prevention of polyspermy at the sperm concentrations normally encountered by the egg.     

Suitability of non-fertilized eggs of Homalodisca vitripennis for the egg parasitoid Gonatocerus morrilli

Gonatocerus morrilli (Howard) (Hymenoptera: Mymaridae) is an egg parasitoid used in California, USA to control glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae). Virgin GWSS females deposit non-fertilized eggs and mated females can exhaust sperm reserves for egg fertilization. However, nothing is known about Gonatocerus spp. performance when using non-fertilized GWSS eggs as hosts. Host age preference for oviposition and suitability of non-fertilized GWSS eggs as hosts for G. morrilli reproduction were investigated to determine whether non-fertilized eggs on sentinel plants could be used to monitor egg parasitoid populations. Gonatocerus morrilli parasitized all ages of GWSS eggs (1–8 days old) regardless if the host egg was fertilized or not. In choice tests (fertilized versus non-fertilized eggs), parasitoids failed to emerge as adults from non-fertilized eggs more often than from fertilized eggs. The results indicate that non-fertilized eggs were accepted by G. morrilli as suitable hosts for oviposition, but were less suitable for immature development compared to fertilized eggs.     

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.     

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.     

Intracellular pH controls the development of new potassium conductance after fertilization of the sea urchin egg

Fertilization of the sea urchin egg leads to a sequence of changes at the egg surface and the interior cytoplasm. Among these changes are the transient elevation of internal calcium levels, alkalization of the cytoplasm and development of new K⁺-conductance. In the series of experiments reported here, we separate the effects on potassium activation of the calcium release and the rise in the intracellular pH. The development of new K⁺-conductance was dependent on alkalization of the egg cytoplasm, and not on a rise of internal calcium levels. The effects of 2,4-dinitrophenol, N-ethylmalemide, antimycin A and oligomycin suggest that the maintenance of the alkaline internal pH of fertilized eggs appears to be dependent on membrane ATPase activity.     

Reconstitution into liposomes of the Na+-dependent transport systems for cyclacillin,an aminopenicillin,from brush border membranes of rat small intestine

Triton X-100 extract from brush border membranes of rat small intestine was recombined with egg phosphatidylcholine liposomes by the freeze-thaw sonication method. The treated liposomes showed a Na⁺-dependent uptake of cyclacillin, which was inhibited by a low concentration of mercuric ions and L-phenylalanylglycine, but not by glycine. These are consistent with the absorption characteristics of the antibiotic in situ and indicate that reconstitution of the Na⁺-dependent active cyclacillin transport system of rat small intestine has been achieved.     

Alterations of PLCbeta1 in mouse eggs change calcium oscillatory behavior following fertilization

Inositol 1,4,5-trisphosphate generated by the action of a phospholipase C (PLC) mediates release of intracellular Ca²⁺ that is essential for sperm-induced activation of mammalian eggs. Much attention currently focuses on the role of sperm-derived PLCζ in generating changes in egg intracellular Ca²⁺ despite the fact that PLCζ constitutes a very small fraction of the total amount of PLC in a fertilized egg. Eggs express several isoforms of PLC, but a role for an egg-derived PLC in sperm-induced Ca²⁺ oscillations has not been examined. Reducing egg PLCβ1 by a transgenic RNAi approach resulted in a significant decrease in Ca²⁺ transient amplitude, but not duration or frequency, following insemination. Furthermore, overexpressing PLCβ1 by microinjecting a Plcb1 cRNA significantly perturbed the duration and frequency of Ca²⁺ transients and disrupted the characteristic shape of the first transient. These results provide the first evidence for a role of an egg-derived PLC acting in conjunction with a sperm-derived PLCζ in egg activation.     

ELECTRIC IMPEDANCE OF ARBACIA EGGS

The alternating current resistance and capacity of suspensions of unfertilized and fertilized eggs of Arbacia punctulata have been measured at frequencies from 10³ to 1.64 x 10⁷ cycles per second. The unfertilized egg has a static plasma membrane capacity of 0.73 µf./cm.² which is practically independent of frequency. The fertilized egg has a static membrane capacity of 3.1 µf./cm.² at low frequencies which decreases to a value of 0.55 µf./cm.² at high frequencies. The decrease follows closely the relaxation dispersion of the dielectric constant if the dissipation of such a system is ignored. It is considered more probable that the effect is due to a fertilization membrane of 3.1 µf./cm.² capacity lifted 1.5 µ. from the plasma membrane, the interspace having the conductivity of sea water. The suspensions show a frequency-dependent capacity at low frequencies which may be attributable to surface conductance. The equivalent low frequency internal specific resistance of both the unfertilized and fertilized egg is about 186 ohm cm. or about 6 times that of sea water, while the high frequency data extrapolate to a value of about 4 times sea water. There is evidence at the highest frequencies that the current is penetrating the nucleus and other materials in the cytoplasm. If this effect were entirely due to the nucleus it would lead to a very approximate value of 0.1 µf./cm.² for the capacity of the nuclear membrane. The measurements do not indicate any change in this effect on fertilization.     

THE BEHAVIOR OF THE NUCLEIC ACIDS DURING THE EARLY DEVELOPMENT OF THE SEA URCHIN EGG (ARBACIA)

The authors wish to correct an error in the paper "The behavior of the nucleic acids during the early development of the sea urchin egg (Arbacia)" (J. Gen. Physiol., 1947–48, 31, 203). Owing to an oversight, the figures for the amounts of various P fractions in a single Arbacia egg have been erroneously expressed in γ x 10^–3 units (Tables I and II, page 205; the last two lines of page 206). The figures should have been expressed in γ x 10^–5 units. Thus, the fertilized Arbacia egg contains an average of 20 γ x 10^–5 ribonucleic acid P and 0.7 to 1 γ x 10^–5 desoxyribonucleic acid P.     

Electrical properties of the Drosophila egg membrane

Electrical properties of the egg membrane of Drosophila melanogaster were examined using intracellular microelectrodes. Unfertilized eggs and fertilized eggs for the period up to the syncytial blastoderm stage were used. Among Na, K, and Cl, K was most permeant to the membrane. The K permeability, however, did not completely determine the membrane potential. The resting potential in standard solution was ?63.5 ± 8.0 mV (mean ± SD) in unfertilized eggs collected within 2–3 days after virgin flies started to lay eggs. The resting potential in fertilized eggs was ?27.0 ± 8.4 mV within 20 min after egg deposition, while it was ?55.1 ± 6.5 mV at the syncytial blastoderm stage. These changes at different developmental stages were associated with changes in the K-dependence of the membrane. The larger amplitude of the resting potential was suggested to be due to increased K permeability but not to decreased nonspecific leakage. The current-voltage relation was linear throughout the stages examined. Action potentials, such as those in eggs of other animals, were not observed. High Ca media significantly increased the amplitude of the resting potential associated with increase in the membrane resistance. A remarkable nonlineality in the I–V relation appeared in high Ca media, which caused continuously increasing hyperpolarization during sustained inward current. Eggs of temperature-sensitive mutants, shi^ts1 and para^ts1 showed properties similar to those in wild-type eggs with transient temperature changes.     

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca²⁺) often termed a ''Ca²⁺ wave''. While the speed and number of oscillations of the Ca²⁺ wave varies between species, the change in intracellular Ca²⁺ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca²⁺-dependent events. Here we present a protocol for imaging the Ca²⁺ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca²⁺ wave and the downstream changes associated with it.     

Synthesis of nucleic acid purines in the sea urchin embryo

Embryos of the sea urchin, Arbacia punctulata, were shown to utilize carboxyl labeled acetate and glycine for the synthesis of nucleic acids and protein.The hypothesis that DNA must arise exclusively from the pre-formed RNA of the virgin egg is unlikely since the specific activities of DNA and its isolated purines from the blastula were at least 10 times greater than the corresponding values for RNA.The specific activities of adenine and guanine of RNA formed from glycine-1-C¹⁴ were identical, whereas in DNA the specific activity of adenine was less than half that of guanine.Comparison of the specific activities of the DNA purines with the glycine in the medium indicated that a large part of the newly formed DNA arose from unlabeled endogenous precursor.     

Cell-cycle dependency of radiosensitivity and mutagenesis in fertilized egg cells of rice,Oryza sativa L.

Summary To determine the time and duration of the first and second DNA synthetic phases in fertilized egg cells and central cells of rice, a total of 753 ovules were sampled at 2 h intervals during the first 30 h after pollination and exposed to ³H-thymidine for 2 h at 25 °C. Autoradiographic observation of labeled nuclei was made for fertilized egg cells, as well as for central and antipodal cells. The first and second DNA synthetic phases in fertilized egg cells were found 8–12 h and 21–25 h after pollination, respectively. The durations of each cell-cycle phase in the egg cell were estimated to be 4–6 h for G₁, 4 h vor S and for G₂, and 2 h for M. In the central cell, the first DNA synthesis took place at 3–4 h after pollination, i.e., immediately after fertilization, followed by the formation of the primary endosperm nucleus. Antipodal cells also showed labeled nuclei in the early stages after fertilization. The first divisions of fertilized egg cell and primary endosperm nucleus were observed at 16–18h and at 4–6 h after pollination, respectively. The present observations suggest that sperm and egg nuclei participate in fertilization with haploid amount (1C) of DNA and fertilized egg cell originates thus in 2C state.     

Effects of EHF radiation and cytoactive substances on fertilization and early embryonic development of the sea urchin Strongylocentrotus intermedius

This study examines the influence of extremely high frequency (EHF) electromagnetic radiation (42.2 GHz, 100 μW/cm², impulse modulation 1000 Hz) on the gametes and embryos of the sea urchin Strongylocentrotus intermedius. Preliminary radiation treatment reduces the number of fertilized egg cells and the survival rate of embryos at early stages of development up to the pluteus. Methyluracil reduces the degree of expression of biological effects of EHF radiation.     

ELECTRIC IMPEDANCE OF THE FROG EGG

Electrical impedance measurements were made upon unfertilized and fertilized eggs of the leopard frog, Rana pipiens, over a frequency range of 0.05 to 10 kc. Average values of 170 ohm cm.² were obtained for the plasma membrane resistance of the egg, 2.0 µf/cm.² for the plasma membrane capacity, 86° for the phase angle of the membrane, and 570 ohm cm. for the specific resistance of the interior. These values did not change upon fertilization. No spontaneous rhythmical impedance changes such as have been found by Hubbard and Rothschild in the trout egg were found in frog eggs.     

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.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.