Increased uptake of thymidine in the activation of sea urchin eggs : Specificity of uptake and dependence on internal pH, the cortical reaction, and external sodium

The uptake of thymidine in sea urchin eggs is considered in terms of its specificity, the cortical reaction, and the increase of intracellular pH following fertilization. The rate of uptake increases greater than 50-fold after fertilization. All deoxyribonucleosides and ribonucleosides tested compete with thymidine for transport sites. Free pyrimidine and purine bases, deoxyribonucleotides, and amino acids do not compete, showing that the specificity of this uptake lies at the nucleoside level. Uptake may be turned on in unfertilized eggs by treatment with ammonia, a treatment known to by-pass the cortical reaction and raise intracellular pH. However, when compared with uptake in fertilized eggs, it proceeds later and at a lower rate. Both of these deficiencies are overcome by fertilizing the ammonia-treated eggs or treating them with butyric acid or ionophore A23187. These treatments induce the cortical reaction and stimulate an immediate and complete turn-on of thymidine uptake. Superseding these apparent involvements of the cortical reaction and mtracellular pH in thymidine uptake is an extremely strict requirement for extracellular Na⁺.     

Requirement of Extracellular Calcium Ions for the Early Fertilization Events in the Medaka Egg

The requirement for calcium and the change in calcium content in eggs of Oryzias iatipes during the cortical reaction and sperm penetration were examined. Naked eggs failed to exhibit the cortical reaction upon insemination under Ca Mg-free conditions. These eggs exhibited the cortical reaction by reinsemination in the presence of extracellular Ca²⁺. The effect of extracellular Ca²⁺ on sperm penetration could be replaced by one of several divalent cations in the external medium. Unlike the cortical reaction, sperm penetration failed to be induced by microinjection to increase intracellular Ca²⁺. Verapamil significantly reduced the action of extracellular Ca²⁺ or Ba²⁺ of divalent cations examined in fertilization, while TEA and TTX had no effect on fertilization in the presence of these cations. No ⁴⁵Ca uptake into the egg proper was recognized before completion of the cortical reaction. These observations suggest that extracellular divalent cations are indispensable for sperm stimulation of the egg and its penetration into the egg, for which an influx of Ca²⁺ from the external medium is not required.     

Induction of the cortical reaction in isolated sea urchin egg cortices: effects of Ca2+ and ionophore A23187

The cortical reaction in isolated sea urchin (Strongylocentrotus purpuratus) egg cortices has been monitored with phase-contrast video microscopy. It was confirmed that the cortical reaction is induced by exposure to Ca2+. No induction was observed after exposure to the Ca2+-ionophore A23187, although the cortices remain sensitive to a subsequent exposure to Ca2+, and the cortical reaction in unfertilized eggs suspended in cortex isolation medium remains inducible by exposure to A23187. These results imply: (1) that A23187 does not induce the cortical reaction directly; (2) that the release of intracellular Ca2+, through which A23187 induces the cortical reaction, is not from storage sites localized entirely in the cortex; and (3) that intracellular storage sites for the Ca2+ involved in the cortical reaction are also present outside the cortex.     

Protein synthesis is required for the transition to Ca(2+)-dependent regulated secretion in progesterone-matured Xenopus oocytes

Calcium (Ca) ionophores trigger cortical granule exocytosis in progesterone-matured Xenopus oocytes (eggs), but not in immature oocytes. Prior work suggested that this secretory transition involved a Ca-dependent isoform of protein kinase C (PKC). To address this possibility, we treated eggs with several different inhibitors of Ca-dependent PKCs. Although these agents (eg., staurosporine, Ro31-8220) completely blocked cortical granule exocytosis that is triggered in eggs by phorbol esters, they had no impact on ionomycin-evoked secretion of cortical granule lectin. These data suggest that Ca-dependent PKCs do not mediate secretory triggering in eggs. Instead, further investigation revealed that protein synthesis (but not RNA synthesis) was required for eggs to secrete in response to ionomycin. Moreover, we observed that when oocytes were matured by injection of maturation promoting factor (MPF), they failed to secrete in response to ionomycin. Collectively, these results suggest that the progesterone-dependent maturation pathway induces these cells either to synthesize de novo, a protein that mediates Ca-dependent secretory triggering, or that intrinsic Ca-sensing machinery is modified in a protein-synthesis-dependent fashion. Initial efforts to distinguish between these possibilities (using Ca overlay, pharmacological and immunoblot strategies) revealed that such Ca-binding proteins as calmodulin, synaptotagmin1, CAPS, rabphilin-3A and calcineurin were unlikely to transduce the secretory effects of ionomycin in eggs. Thus, the cortical reaction in these cells may rely on a novel mechanism for initiating Ca-dependent exocytosis.     

The cortical endoplasmic reticulum (ER) of the mouse egg: localization of ER clusters in relation to the generation of repetitive calcium waves.

The endoplasmic reticulum (ER) of the mature mouse egg consists of a fine tubular network and pronounced accumulations in the cortex. The ER was visualized both in intact eggs and with in vitro preparations of the cortex using the fluorescent lipophilic dye, DiI. Immunofluorescent labeling of the ER in isolated cortical preparations demonstrated that the ER clusters contain inositol 1,4, 5-trisphosphate (IP(3)) receptors, indicating an important involvement in sperm-induced Ca(2+) transients, which are triggered by IP(3). We imaged the ER during fertilization and the subsequent Ca(2+) transients and found that the clusters remained intact throughout this period. Recovery of fluorescence after photobleaching established that the ER clusters are continuous with the reticular ER network and that these structures remain stable and continuous throughout the time of fertilization-induced Ca(2+) transients; continuity also remained during IP(3) injection. These results indicate that, in contrast to echinoderm eggs, the ER of mouse eggs does not become disrupted when it releases Ca(2+)at fertilization. The localization and apparent stability of the cortical ER clusters may be important in generating Ca(2+) oscillations, which are characteristic of fertilized mammalian eggs. Imaging of intracellular Ca(2+) revealed that Ca(2+) transients originate in the hemisphere of the egg that contains abundant ER clusters, thus the mouse contains a stable cortical pacemaker responsible for generating Ca(2+) waves.     

Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus laevis.

Transit into interphase of the first mitotic cell cycle in amphibian eggs is a process referred to as activation and is accompanied by an increase in intracellular free calcium [( Ca2+]i), which may be transduced into cytoplasmic events characteristic of interphase by protein kinase C (PKC). To investigate the respective roles of [Ca2+]i and PKC in Xenopus laevis egg activation, the calcium signal was blocked by microinjection of the calcium chelator BAPTA, or the activity of PKC was blocked by PKC inhibitors sphingosine or H7. Eggs were then challenged for activation by treatment with either calcium ionophore A23187 or the PKC activator PMA. BAPTA prevented cortical contraction, cortical granule exocytosis, and cleavage furrow formation in eggs challenged with A23187 but not with PMA. In contrast, sphingosine and H7 inhibited cortical granule exocytosis, cortical contraction, and cleavage furrow formation in eggs challenged with either A23187 or PMA. Measurement of egg [Ca2+]i with calcium-sensitive electrodes demonstrated that PMA treatment does not increase egg [Ca2+]i in BAPTA-injected eggs. Further, PMA does not increase [Ca2+]i in eggs that have not been injected with BAPTA. These results show that PKC acts downstream of the [Ca2+]i increase to induce cytoplasmic events of the first Xenopus mitotic cell cycle.     

Induction of calcium-dependent, localized cortical granule breakdown in sea-urchin eggs by voltage pulsation

A technique that employs a high-voltage pulses to produce pores in cell membranes (Kinosita and Tsong (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1923) has been used to investigate the role of Ca2+ in the early events of activation of sea-urchin eggs. Exposure of eggs to a voltage pulse of 1 kV/cm for 100 microseconds resulted in localized exocytosis of the contents of cortical granules and development of a partial fertilization envelope. This effect was triggered by entrance of Ca2+ through the voltage-induced pores. In a medium containing 100 microM Ca2+ and 45Ca2+ tracer, the voltage-treated eggs admitted 3.6 +/- 0.3 fmol Ca2+/egg within a few seconds. Untreated eggs took up only 1.0 +/- 0.2 fmol/egg after minutes of incubation. Furthermore, depletion of Ca2+ or the presence of EGTA in the external medium prevented elevation of the fertilization envelope by the voltage pulsation. Delay in Ca2+ addition after the voltage pulsation reduced the fraction of eggs that developed partial fertilization envelope. Loss of essential cytoplasmic components during the delay period is judged unlikely, since these eggs were viable, could form partial fertilization envelopes if re-pulsed in the presence of Ca2+, and could develop to normal blastula stage embryos upon fertilization with sperm. Thus, we interpret this effect as due to a resealing of pores; the half-life of pores being 20 s. The elevation of partial fertilization envelopes occurred only at the loci facing the anode, and multiple pulses with mixing resulted in the formation of multiple fertilization envelopes. These envelopes were stable for up to several hours; further propagation (wave spreading) was not observed. The above results indicate that a primary reaction in the sequence of steps in fertilization envelope formation involves Ca2+ to trigger cortical granule breakdown and formation of the fertilization envelope.     

"Cleavage" and cortical granule breakdown in Rana pipiens oocytes induced by direct microinjection of calcium.

Microinjection of approximately 0.3 mug of calcium into maturing oocytes of Rana pipiens after nuclear dissolution resulted in cleavage-like constrictions, cortical granule breakdown, and formation of a structure resembling a two-cell embryo. Mg2+, Na+, or K+ did not induce any of these reactions. Larger amounts of Ca2+-induced contraction over the entire surface of oocytes or eggs, but did not induce cleavage-like constrictions; smaller amounts of Ca2+ produced either a local cortical granule reaction of the formation of one large and one small "blastomere." Furrow formation was not observed during normally induced maturation until after germinal vesicle breakdown. The location of microinjected Ca2+ determined the orientation of the resulting furrow. Ca2+-induced cortical granule breakdown occurred in full-grown nonmaturing oocytes near the site of injection. Cortical granule breakdown also occurred in maturing oocytes (after germinal vesicle breakdown but before second meiotic metaphase), but only in the blastomere containing the infected Ca2+. As expected, in mature oocytes (at second meiotic metaphase) cortical granule breakdown occurred over the entire oocyte surface, including both blastomeres. The results indicate that furrow formation and cleavage-like constrictions may be directly influenced by Ca2+, and that functional contractile elements are present near all areas of the oocyte surface. Furthermore, Ca2+ injection initiates localized cortical granule breakdown in full-grown immature and maturing oocytes.     

Independent Initiation of Calcium Dependent Glycosidase Release and Cortical Contractions during the Activation of Ascidian Eggs

During fertilization or ionophore induced activation, ascidian eggs rapidly release cell surface N-acetylglucosaminidase activity used in the block against polyspermy and undergo cortical contractions before they re-initiate meiosis. To better understand the activation process, we probed the relationship between these two processes in Ascidia ceratodes eggs by activating with different agents that increase intracellular Ca levels and under different ionic conditions. Glycosidase activity release was followed by the use of a fluorogenic substrate, and cortical contractions were followed by examining changes in cell shape with light microscopy. Ionomycin (2.7 μM) and thimerosal (1 mM) initiate glycosidase release and cortical contractions when administered in complete sea water (SW) but only the contractions in low Ca SW. Ryanodine (0.67 mM), known to raise free intracellular Ca in a number of cell types by release from the endoplasmic reticulum, causes glycosidase release but fails to initiate cortical contractions in complete SW. Thapsigargin (10 μM), which inhibits Ca dependent ATPase in the ER, causes glycosidase release but induces the contractions only about 50% of the time. These experiments show that, although glycosidase release normally precedes the ooplasmic shape changes that accompany the resumption of meiosis in ascidian eggs, they are not obligately coupled. That both processes can be induced by treatments known to raise intracellular Ca in other systems but under different conditions indicates that there may be a multiplicity of Ca requiring but functionally independent events during egg activation.     

Egg-to-embryo transition is driven by differential responses to Ca(2+) oscillation number

Ca(2+) oscillations and signaling represent a basic mechanism for controlling many cellular events. Activation of mouse eggs entrains a temporal series of Ca(2+)-dependent events that include cortical granule exocytosis, cell cycle resumption with concomitant decreases in MPF and MAP kinase activities, and recruitment of maternal mRNAs. The outcome is a switch in cellular differentiation, i.e., the conversion of the egg into the zygote. By activating mouse eggs with experimentally controlled and precisely defined Ca(2+) transients, we demonstrate that each of these events is initiated by a different number of Ca(2+) transients, while their completion requires a greater number of Ca(2+) transients than for their initiation. This combination of differential responses to the number of Ca(2+) transients provides strong evidence that a single Ca(2+) transient-driven signaling system can initiate and drive a cell into a new developmental pathway, as well as can account for the temporal sequence of cellular changes associated with early development.     

Comparison of Ca2+ mobilizing activities of cyclic ADP-ribose and inositol trisphosphate.

We have previously shown that a metabolite of NAD+ generated by an enzyme present in sea urchin eggs and mammalian tissues can mobilize intracellular Ca2+ in the eggs. Structural determination established it to be a cyclized ADP-ribose, and the name cyclic ADP-ribose (cADPR) has been proposed. In this study, Ca2+ mobilizations induced by cADPR and inositol trisphosphate (IP3) in sea urchin egg homogenates were monitored with Ca2+ indicators and Ca2(+)-specific electrodes. Both methods showed that cADPR can release Ca2+ from egg homogenates. Evidence indicated that it did not act as a nonspecific Ca2(+)-ionophore or as a blocker of the microsomal Ca2(+)-transport; instead, it was likely to be operating through a specific receptor system. This was supported by its half-maximal effective concentration of 18 nM, which was 7 times lower than that of IP3. The receptor for cADPR appeared to be different from that of IP3 because heparin, an inhibitor of IP3 binding, had no effect on the cADPR action. The Ca2+ releases induced by cADPR and IP3 were not additive and had an inverse relationship, indicating overlapping stores were mobilized. Microinjection of cADPR into intact eggs induced transient intracellular Ca2+ changes and activated the cortical reaction. The in vivo effectiveness of cADPR was directly comparable with IP3 and neither required external Ca2+. In addition, both were effective in activating the eggs to undergo multiple nuclear cycles and DNA synthesis. These results suggest that cADPR could function as a second messenger in sea urchin eggs.     

Microinjection of cyclic ADP-ribose triggers a regenerative wave of Ca2+ release and exocytosis of cortical alveoli in medaka eggs

Medaka (Oryzias latipes) eggs microinjected with the Ca(2+)-mobilising messenger cyclic adenosine diphosphate ribose (cADPR) underwent a wave of exocytosis of cortical alveoli and were thus activated. The number of eggs activated was sharply dependent on the concentration of cADPR in the pipette, the threshold concentration was approximately 60 nM. After injection, a pronounced latency preceded the onset of cortical alveoli exocytosis; this latency was independent of the concentration of cADPR but decreased markedly with increasing temperature. Heat-treated cADPR, which yields the inert non-cyclised product ADP-ribose, was ineffective in activating eggs. When cADPR was injected into aequorin-loaded eggs, a wave of luminescence arose at the site of cADPR injection and then swept out across the egg with a mean velocity of approximately 13 microns/s; the velocity was independent of the concentration of injected cADPR. In such a large cell (diameter of around 1 mm), this is considerably faster than that possible by simple diffusion of cADPR, which unambiguously demonstrates that cADPR must activate a regenerative process. cADPR has been demonstrated to modulate Ca(2+)-induced Ca2+ release (CICR) via ryanodine receptors (RyRs) in many cell types, and consistent with this was the finding that microinjection of the pharmacological RyR modulator, ryanodine, also activated medaka eggs. These results suggest that a cADPR-sensitive Ca2+ release mechanism is present in the medaka egg, that cADPR is the most potent activator of medaka eggs described to date, and that it activates eggs by triggering a wave of CICR from internal stores that in turn stimulates a wave of exocytosis.     

The GTP-binding protein RhoA localizes to the cortical granules of Strongylocentrotus purpuratas sea urchin egg and is secreted during fertilization

The sea urchin egg has thousands of secretory vesicles known as cortical granules. Upon fertilization, these vesicles undergo a Ca2+-dependent exocytosis. G-protein-linked mechanisms may take place during the egg activation. In somatic cells from mammals, GTP-binding proteins of the Rho family regulate a number of cellular processes, including organization of the actin cytoskeleton. We report here that a crude membrane fraction from homogenates of Strongylocentrotus purpuratus sea urchin eggs, incubated with C3 (which ADP-ribosylates specifically Rho proteins) and [32P]NAD, displayed an [32P]ADP-ribosylated protein of 25 kDa that had the following characteristics: i) identical electrophoretic mobility in SDS-PAGE gels as the [32P]ADP-ribosylated Rho from sea urchin sperm; ii) identical mobility in isoelectro focusing gels as human RhoA; iii) positive cross-reactivity by immunoblotting with an antibody against mammalian RhoA. Thus, unfertilized S. purpuratus eggs contain a mammalian RhoA-like protein. Immunocytochemical analyses indicated that RhoA was localized preferentially to the cortical granules; this was confirmed by experiments of [32P]ADP-ribosylation with C3 in isolated cortical granules. Rho was secreted and retained in the fertilization membrane after insemination or activation with A23187. It was observed that the Rho protein present in the sea urchin sperm acrosome was also secreted during the exocytotic acrosome reaction. Thus, Rho could participate in those processes related to the cortical granules, i.e., in the Ca2+-regulated exocytosis or actin reorganization that accompany the egg activation.     

Effects of egg quality on normal fertilization and early development of the cod, Gadus morhua L

Normal cod eggs respond to insemination by a rapid cortical reaction followed by an increase in total osmolarity and a small increase in egg diameter. The chorion becomes harder, but this is a slower process reaching its maximum strength after c . 24 h. Bad eggs are characterized by a slower or incomplete cortical reaction, resulting in a slower rise in osmolarity and a softer chorion. Bad eggs rapidly lose their capacity for fertilization. In unfertilized eggs in sea water, no cortical reaction is observed. There is, however, a rise in total osmolarity and a hardening of the chorion.     

The effect of nicotine on sperm-Egg interaction in the sea urchin: Polyspermy and electrical events

We have studied some of the effects of nicotine on sea urchin eggs, spermatozoa, and their interaction using electrical recording techniques and fertilization-rate experiments. Pretreating eggs with nicotine enhances the fertilization rate, whereas this drug has an inhibitory effect on spermatozoa. Pulse-treated eggs or eggs fertilized in the presence of nicotine give rise to attenuated step depolarizations, which may be attributed to a decrease in membrane resistance (Rm) of the egg or, in the latter case, to an alteration to the spermatozoon. Concurrently, with the change in the step depolarization there is a reduction in amplitude of the fertilization potential (FP) suggesting that the cortical reaction is in some way altered. Nicotine has no effect on the Rm of fertilized eggs or oocytes, where there are no cortical granules. We suggest that nicotine alters the cortex of sea urchin eggs–possibly by causing a partial dissolution of cortical granules–which renders the eggs more receptive to spermatozoa. The reductions in amplitude of the step depolarization and the FP are consequences of this alteration.     

Mastoparan induces Ca2+-independent cortical granule exocytosis in sea urchin eggs

In most species, cortical granule exocytosis is characteristic of egg activation by sperm. It is a Ca(2+)-mediated event which results in elevation of the vitelline coat to block permanently the polyspermy at fertilization. We examined the effect of mastoparan, an activator of G-proteins, on the sea urchin egg activation. Mastoparan was able to induce, in a concentration-dependent manner, the egg cortical granule exocytosis; mastoparan-17, an inactive analogue of mastoparan, had no effect. Mastoparan, but not sperm, induced cortical granule exocytosis in eggs preloaded with BAPTA, a Ca(2+) chelator. In isolated egg cortical lawns, which are vitelline layers and membrane fragments with endogenously docked cortical granules, mastoparan induced cortical granule fusion in a Ca(2+)-independent manner. By contrast, mastoparan-17 did not trigger fusion. We conclude that in sea urchin eggs mastoparan stimulates exocytosis at a Ca(2+)-independent late site of the signaling pathway that culminates in cortical granule discharge.     

An ultrastructural study of cross-fertilization (Arbacia female x Mytilus male)

Insemination of sea urchin (Arbacia) ova with mussel (Mytilus) sperm has been accomplished by treating eggs with trypsin and suspending the gametes in seawater made alkaline with NaOH. Not all inseminated eggs undergo a cortical granule reaction. Some eggs either elevate what remains of their vitelline layer or demonstrate no cortical modification whatsoever. After its incorporation into the egg, the nucleus of Mytilus sperm undergoes changes which eventually give rise to the formation of a male pronucleus. Concomitant with these transformations, a sperm aster may develop in association with the centrioles brought into the egg with the spermatozoon. Both the male pronucleus and the sperm aster may then migrate centrad to the female pronucleus. Evidence is presented which suggests that fusion of the male pronuclei from Mytilus sperm with female pronuclei from Arbacia eggs may occur, although this was not directly observed. These results demonstrate that Mytilus sperm nuclei are able to react to conditions within Arbacia eggs and differentiate into male pronuclei.     

Maitotoxin triggers the cortical reaction and phosphatidylinositol-4,5-bisphosphate breakdown in amphibian oocytes

Maitotoxin, a potent marine toxin extracted from peredinians, was found to mimic fertilization in Xenopus oocytes and to trigger the breakdown of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2, the precursor of inositol 1,4,5-trisphosphate], an increase of intracellular pCa and the cortical reaction, including the exocytosis of cortical granules and a wave-like propagation of contraction in the animal hemisphere. All these effects of maitotoxin required the presence of external calcium. Moreover, the toxin considerably increased Ca2+ influx in amphibian oocytes arrested at first meiotic prophase, due to the permanent activation of voltage-dependent Ca2+ channels. Nevertheless it is doubtful that maitotoxin acts primarily as a Ca2+ ionophore or at the level of Ca2+ channels. Indeed no stimulation of Ca2+ uptake was observed in metaphase-II-arrested oocytes, although maitotoxin readily triggered the breakdown of PtdIns(4,5)P2 as well as the cortical reaction in such cells. On the other hand, PtdIns(4,5)P2 breakdown was not reduced in oocytes microinjected with EGTA, although the calcium chelator prevented the oocytes from undergoing the cortical reaction. Taken together, these findings support the view that the toxin might act primarily by increasing PtdIns(4,5)P2 phosphodiesterase activity.     

Excitotoxic injury to mitochondria isolated from cultured neurons

Neuronal death in response to excitotoxic levels of glutamate is dependent upon mitochondrial Ca2+ accumulation and is associated with a drop in ATP levels and a loss in ionic homeostasis. Yet the mapping of temporal events in mitochondria subsequent to Ca2+ sequestration is incomplete. By isolating mitochondria from primary cultures, we discovered that glutamate treatment of cortical neurons for 10 min caused 44% inhibition of ADP-stimulated respiration, whereas the maximal rate of electron transport (uncoupler-stimulated respiration) was inhibited by approximately 10%. The Ca2+ load in mitochondria from glutamate-treated neurons was estimated to be 167 +/- 19 nmol/mg protein. The glutamate-induced Ca2+ load was less than the maximal Ca2+ uptake capacity of the mitochondria determined in vitro (363 +/- 35 nmol/mg protein). Comparatively, mitochondria isolated from cerebellar granule cells demonstrated a higher Ca2+ uptake capacity (686 +/- 71 nmol/mg protein) than the cortical mitochondria, and the glutamate-induced load of Ca2+ was a smaller percentage of the maximal Ca2+ uptake capacity. Thus, this study indicated that Ca(2+)-induced impairment of mitochondrial ATP production is an early event in the excitotoxic cascade that may contribute to decreased cellular ATP and loss of ionic homeostasis that precede commitment to neuronal death.