Evidence for the involvement of a Src-related tyrosine kinase in Xenopus egg activation.

Recently, we have purified a Src-related tyrosine kinase, named Xenopus tyrosine kinase (Xyk), from oocytes of Xenopus laevis and found that the enzyme is activated within 1 min following fertilization [Sato et al. (1996) J. Biol. Chem. 271, 13250-13257]. A concomitant translocation of a part of the activated enzyme from the membrane fraction to the cytosolic fraction was also observed. In the present study, we show that parthenogenetic egg activation by a synthetic RGDS peptide [Y. Iwao and T. Fujimura, T. (1996) Dev. Biol. 177, 558-567], an integrin-interacting peptide, but not by electrical shock or the calcium ionophore A23187 causes the kinase activation, tyrosine phosphorylation, and translocation of Xyk. A synthetic tyrosine kinase-specific inhibitor peptide was employed to analyze the importance of the Xyk activity in egg activation. We found that the peptide inhibits the kinase activity of purified Xyk at IC50 of 8 microM. Further, egg activation induced by sperm or RGDS peptide but not by A23187 was inhibited by microinjection of the peptide. In the peptide-microinjected eggs, penetration of the sperm nucleus into the egg cytoplasm and meiotic resumption in the egg were blocked. Indirect immunofluorescence study demonstrates that Xyk is exclusively localized to the cortex of Xenopus eggs, indicating that Xyk can function in close proximity to the sperm-egg or RGDS peptide-egg interaction site. Taken together, these data suggest that the tyrosine kinase Xyk plays an important role in the early events of Xenopus egg activation in a manner independent or upstream of calcium signaling.     

Protein tyrosine kinase activity of eggs of the sea urchin Strongylocentrotus purpuratus: the regulation of its increase after fertilization

Protein tyrosine kinase activity in eggs of the sea urchin, Strongylocentrotus purpuratus, increased two- to fourfold as early as several min after fertilization at 8-10 degrees C. Artificial activation of eggs with the divalent cation ionophore, A23187, or with butyric acid induced the increase in enzyme activity. The transfer of eggs to seawater containing either no Na+ or 50 mM Na+ and 10(-4) M amiloride immediately after fertilization did not block the increases in enzyme activity. When eggs were activated with seawater containing NH4OH, enzyme activity did not increase at 1 hr after activation, although the increased activity was detected at 3 hr after activation. Increased enzyme activity also was observed in enucleated egg fragments activated with butyric acid. Puromycin and emetine, inhibitors of protein synthesis, also did not inhibit the initial increases of enzyme activity after fertilization. These results demonstrated that the increased protein tyrosine kinase activity observed after fertilization of S. purpuratus eggs can be initiated independent of various other known events such as fusion with sperm cells and protein and DNA synthesis.     

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.     

Tyrosine kinase-dependent activation of phospholipase Cgamma is required for calcium transient in Xenopus egg fertilization

In a previous study (K.-I. Sato et al., 1999, Dev. Biol. 209, 308-320), we presented evidence that a Src-related protein-tyrosine kinase (PTK), named Xyk, may act upstream of the calcium release in fertilization of the Xenopus egg. In the present study, we examined whether PTK activation of phospholipase Cgamma (PLCgamma) plays a role in the fertilization-induced calcium signaling. Immunoprecipitation studies show that Xenopus egg PLCgamma is tyrosine phosphorylated and activated within a few minutes after fertilization but not after A23187-induced egg activation. Consistently, we observed a fertilization-induced association of PLCgamma with Xyk activity that was not seen in A23187-activated eggs. A Src-specific PTK inhibitor, PP1, blocked effectively the fertilization-induced association of PLCgamma with Xyk activity and up-regulation of PLCgamma, when microinjected into the egg. In addition, a PLC inhibitor, U-73122, inhibited sperm-induced inositol 1,4,5-trisphosphate production and the calcium transient and subsequent calcium-dependent events such as cortical contraction, elevation of fertilization envelope, and tyrosine dephosphorylation of p42 MAP kinase, all of which were also inhibited by PP1. On the other hand, A23187 could cause the calcium response and calcium-dependent events in eggs injected with PP1 or U-73122. These results support the idea that Xenopus egg fertilization requires Src-family PTK-dependent PLCgamma activity that acts upstream of the calcium-dependent signaling pathway.     

A 58-kDa Shc protein is present in Xenopus eggs and is phosphorylated on tyrosine residues upon egg activation.

A 58-kDa protein was detected in Xenopus egg lysate by SDS-PAGE and immunoblotting with an antibody raised against adaptor protein Shc, a well known tyrosine kinase substrate in numerous biological events. Tyrosine phosphorylation of the Xenopus Shc protein (p58 xShc) was found to increase 2.3 +/- 0.4-fold (n = 3) upon fertilization. Pretreatment of eggs with the tyrosine kinase inhibitor genistein effectively blocked the fertilization-dependent phosphorylation. Tyrosine phosphorylation of p58 xShc was also observed when eggs were activated parthenogenetically by an integrin-interacting RGDS-peptide which is known to cause egg activation accompanied by intracellular calcium release. On the other hand, other egg-activating treatments such as electrical shock and calcium ionophore, which directly induce the elevation of intracellular calcium, did not show such an effect. It is also suggested that the phosphorylated p58 xShc may play a role unique to the egg activation process because we found that there was no increase of Shc-Grb2 complex after fertilization. These results demonstrate that p58 xShc is a substrate of egg tyrosine kinases which may be activated by sperm-egg interaction and suggest that the phosphorylated p58 xShc may act upstream of the calcium-dependent pathway of egg activation.     

The activation of sea urchin eggs by the divalent ionophores A23187 and X-537A

The divalent ionophores A23187 and X-537A induce parthenogenesis in sea urchin eggs. This results from their ability to mobilize intracellular Ca²⁺, which is implicated in both artificial parthenogenesis as well as the natural fertilization process. A23187 causes expulsion of cortical granules and elevation of the fertilization membrane within 0.5–9 min followed by an initiation of cell cleavage. The broader spectrum ionophore X-537A is less potent, but the production of cytoplasmic aberrations are more apparent. In contrast to the sperm-activated egg, the initial phase of ionophore induced activation is accompanied either by relatively insignificant changes in membrane resistance, or an increase.     

Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg.

The role of calcium in cortical granule exocytosis and activation of the cell cycle at fertilization was examined in the mouse egg using the calcium chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and the fluorescent calcium indicator fluo-3. BAPTA and fluo-3 were introduced into zona-free mouse eggs by a 30-min incubation with 0.01-50 microM BAPTA acetoxymethyl ester (AM) and/or 1-20 microM fluo-3 AM prior to in vitro fertilization. Incubation of eggs in greater than or equal to 5.0 microM BAPTA AM inhibited cortical granule exocytosis in all cases. Introduction of the calcium chelator into the egg blocked second polar body formation at greater than or equal to 1.0 microM BAPTA AM. Sperm entry occurred in all eggs regardless of the BAPTA AM concentration. Sperm induce a large transient increase in calcium lasting 2.3 +/- 0.6 min, followed by repetitive transients lasting 0.5 +/- 0.1 min and occurring at 3.4 +/- 1.4-min intervals. Incubation with greater than or equal to 5.0 microM BAPTA AM inhibited all calcium transients. Introduction of BAPTA also inhibited calcium transients, exocytosis, and the resumption of meiosis following application of the calcium ionophore A23187 or SrCl2, which activate eggs. These results demonstrate that the calcium increase at fertilization is required for cortical granule exocytosis and resumption of the cell cycle in a mammalian egg.     

Ca2+-ionophore-induced microvilli and cortical contractions inXenopus eggs. Evidence for involvement of actomyosin

Summary Using scanning electron microscopy, we show that the calcium ionophore A23187 has a profound effect on the surface morphology ofXenopus laevis eggs. The response to ionophore can be interpreted with respect to animal/vegetal polarity and the presence of an asymmetrically organized actomyosin-based contractile system in the egg cortex. When incubated in ionophore, the egg cortex contracts, pigment granules move towards the animal pole, and microvilli increase dramatically in size. While at first overall microvilli density decreases, many additional microvilli appear later in the animal hemisphere but not in the vegetal hemisphere. Eggs incubated in high concentrations of A23187 undergo the same surface changes at a faster rate, and rupture due to a massive cortical contraction. Local application of ionophore to the egg surface results in increased microvilli size and density in that area, with the animal hemisphere showing the greatest response. Since the effects of ionophore are inhibited by the actomyosin probe, N-ethylmaleimide-modified heavy meromyosin, actomyosin is implicated in the ionophore-induced surface changes.     

Protein kinase C is required for the disappearance of MPF upon artificial activation in mouse eggs

The aim of the present study was to investigate the implication of protein kinase C (PKC) in the mouse egg activation process. We used OAG (1-oleoyl-2-acetyl-sn-glycerol) as a PKC activator, calphostin C as a specific PKC inhibitor, and the calcium ionophore A23187 as a standard parthenogenetic agent. The exposure of zona-free eggs to 150 μM or 50 μM OAG for 10 min resulted in meiosis II completion in ∼80% of instances. By contrast, at a lower concentration (25 μM), the PKC stimulator was ineffective as parthenogenetic agent. Shortly after the application of 150 μM OAG, the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) increased transiently in all the eggs examined, whereas after the addition of 50 μM OAG, [Ca²⁺]_i remained unchanged for at least 20 min. During this period, the activity of M-phase promoting factor (MPF) dramatically decreased and most of the eggs entered anaphase except when the PKC was inhibited by calphostin C. Similarly, MPF inactivation and meiosis resumption were prevented in calphostin C-loaded eggs following treatment with A23187, even though the ionophore-induced Ca²⁺ signalling was not affected. Taken together, our results indicate that stimulation of PKC is a sufficient and necessary event to induce meiosis resumption in mouse eggs and strongly suggest that, in this species, the mechanism by which a transient calcium burst triggers MPF inactivation involves a PKC-dependent pathway. Mol. Reprod. Dev. 48:292–299, 1997. © 1997 Wiley-Liss, Inc.     

Calcium-mediated inactivation of the MAP kinase pathway in sea urchin eggs at fertilization.

We have evaluated the regulation of a 43-kDa MAP kinase in sea urchin eggs. Both MAP kinase and MEK (MAP kinase kinase) are phosphorylated and active in unfertilized eggs while both are dephosphorylated and inactivated after fertilization, although with distinct kinetics. Reactivation of MEK or the 43-kDa MAP kinase prior to or during the first cell division was not detected. Confocal immunolocalization microscopy revealed that phosphorylated (active) MAP kinase is present primarily in the nucleus of the unfertilized egg, with some of the phosphorylated form in the cytoplasm as well. Incubation of unfertilized eggs in the MEK inhibitor U0126 (0.5 microM) resulted in the inactivation of MEK and MAP kinase within 30 min. Incubation in low concentrations of U0126 (sufficient to inactivate MEK and MAP kinase) after fertilization had no effect on progression through the embryonic cell cycle. Microinjection of active mammalian MAP kinase phosphatase (MKP-3) resulted in inactivation of MAP kinase in unfertilized eggs, as did addition of MKP-3 to lysates of unfertilized eggs. Incubation of unfertilized eggs in the Ca(2+) ionophore A23187 led to inactivation of MEK and MAP kinase with the same kinetics as observed with sperm-induced egg activation. This suggests that calcium may be deactivating MEK and/or activating a MAP kinase-directed phosphatase. A cell-free system was used to evaluate the activation of phosphatase separately from MEK inactivation. Unfertilized egg lysates were treated with U0126 to inactivate MEK and then Ca(2+) was added. This resulted in increased MAP kinase phosphatase activity. Therefore, MAP kinase inactivation at fertilization in sea urchin eggs likely is the result of a combination of MEK inactivation and phosphatase activation that are directly or indirectly responsive to Ca(2+).     

Activation of Xenopus Eggs by the Kinase Inhibitor 6-DMAP Suggests a Differential Regulation of Cyclin B and p39 Proteolysis

In Xenopus eggs, metaphase II arrest is due to the cytostatic factor that maintains a high level of MPF activity. Kinases are important in this phenomenon since p39^mos and MAPK play a part in the cytostatic activity whereas p34^cdc2 is the catalytic subunit of MPF. Fertilization induces a rise in intracellular calcium leading to egg activation that can be mimicked by calcium-increasing agents such as calcium ionophore. We have performed on Xenopus eggs a biochemical comparison of the effects of the kinase inhibitor 6-DMAP and the calcium ionophore. Both drugs were able to induce pronucleus formation but the underlying molecular events were different. The inactivation of MAPK occurred earlier in eggs exposed to 6-DMAP. Cyclins B1 and B2 were stable and p39^mos was proteolysed in 6-DMAP-treated eggs while the three proteins underwent degradation in A23187-treated ones. These results suggest a differential regulation of ubiquitin-dependent proteolysis of cyclin B and p39^mos.     

Evidence that a starfish egg Src family tyrosine kinase associates with PLC-gamma1 SH2 domains at fertilization

The initiation of calcium release at fertilization in the eggs of most animals relies on the production of IP3, implicating the activation of phospholipase C. Recent work has demonstrated that injection of PLC-gamma SH2 domain fusion proteins into starfish eggs specifically inhibits the initiation of calcium release in response to sperm, indicating that PLC-gamma is necessary for Ca2+ release at fertilization [Carroll et al. (1997) J. Cell Biol. 138, 1303-1311]. Here we investigate how PLC-gamma may be activated, by using the PLC-gamma SH2 domain fusion protein as an affinity matrix to identify interacting proteins. A tyrosine kinase activity and an egg protein of ca. Mr 58 K that is recognized by an antibody directed against Src family tyrosine kinases associate with PLC-gamma SH2 domains in a fertilization-dependent manner. These associations are detected by 15 s postfertilization, consistent with a function in releasing Ca2+. Calcium ionophore treatment of eggs did not cause association of the kinase activity or of the Src family protein with the PLC-gamma SH2 domains. These data identify an egg Src family tyrosine kinase as a potential upstream regulator of PLC-gamma in the activation of starfish eggs.     

Temporal and spatial correlation of fertilization current, calcium waves and cytoplasmic contraction in eggs of Ciona intestinalis

Eggs of the ascidian Ciona intestinalis were loaded with the calcium indicator fura-2 via whole-cell clamp electrodes and changes in cytoplasmic calcium and cell currents were monitored during fertilization either in separate eggs or simultaneously in the same egg. The first indication of egg activation was the fertilization current; which reached peak values around 1 nA after 30 s. A wave of elevated calcium was detectable between 5 s and 30 s (mean = 21 s) after the start of the fertilization current. This wave spread across the egg increasing cytoplasmic calcium levels to at least 10 microM. When the fertilization current and calcium wave were complete and cytoplasmic calcium levels were decreasing to prefertilization levels, a cortical contraction wave spread across the egg surface. In eggs showing normal fertilization current, the calcium wave and the contraction wave were in the same direction. A region of elevated calcium persisted at the animal pole. Changing cytoplasmic calcium levels locally by local application of ionophore A23187 caused a contraction wave originating at the site of ionophore application. Increasing cytoplasmic calcium uniformly by facilitating calcium entry through voltage-regulated channels did not result in a contraction wave.     

Adenosine receptor activation in quiescent Swiss 3T3 cells. Enhancement of cAMP levels, DNA synthesis and cell division

Anti-tubulin immunofluorescence microscopy is used here to demonstrate that eggs of Lytechinus variegatus are induced to assemble cytoplasmic microtubules upon artificial activation. These microtubules progress through three distinct configurations followed by cycles of abortive division. The first of these is a configuration in which microtubules are found in a disordered network near the egg cortex; the progressive thickening of the microtubule-containing layer appears to be responsible for the centripetal movement of the egg nucleus that occurs shortly after activation. These microtubules are replaced at about 40 min by a population of long, radially arrayed microtubules, which are restructured by about 70 min to form the apolar mitotic apparatus. Each of the microtubule configurations characteristic of activated eggs becomes more prominent when eggs are treated at the appropriate times after activation with the microtubule-stabilizing drug taxol. Any microtubule organizing centers within the activated egg must have very limited authority, since aster-like structures are not seen, and microtubules are not observed to be closely associated with the nucleus or egg cortex. Activation of eggs with ammonia in Ca²⁺-free sea water (a treatment that bypasses the cortical reaction and the Ca²⁺ transient) induces the appearance of microtubules as readily and in the same patterns as does treatment with ionophore A23187 or butyric acid, both of which activate by inducing an intracellular calcium release and the cortical reaction.     

Involvement of protein kinase C in translocation of desmoplakins from cytosol to plasma membrane during desmosome formation in human squamous cell carcinoma cells grown in low to normal calcium concentration

The intracellular signal transduction mechanism leading to desmosome formation in low-calcium-grown keratinocytes after addition of calcium to the medium was studied by immunofluorescence using antibodies to desmoplakins I and II (cytoplasmic desmosomal proteins) and by electron microscopy before and after addition of calcium; protein kinase C (PKC) activators 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-dibutyrate (PDBu), and 1,2-dioctanoylglycerol (DOG); calcium ionophore A23187; selective PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine; and a Ca2+/calmodulin-dependent kinase inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). In previous studies using a low-calcium-grown human epidermal squamous cell carcinoma, we have shown that an increase in extracellular Ca2+ caused a four-fold increase in PKC activity and addition of TPA (10 ng/ml) induced a transient increase in membrane-bound PKC activity in association with cell-cell contact formation. The present study showed that TPA (10 ng/ml). PDBu (10 ng/ml), and DOG (1 mg/ml) induced a rapid cell-cell contact and redistribution of desmoplakins from cytoplasm to the plasma membrane with desmosome formation within 60-120 min, which was similar, although less marked, to the effect of increased Ca2+. The TPA-induced desmosome formation was inhibited by selective PKC inhibitors, H-7 (20 microM) or staurosporine (100 nM). On the other hand, calcium ionophore A23187 induced only a temporary increase in the number of desmoplakin-containing fluorescent spots in the cytoplasm and a temporary cell-cell attachment without desmosome formation. The calcium-induced desmosome formation was partially inhibited by 20-100 microM H-7 or 100 nM staurosporine; however, it was not inhibited by W-7 at a concentration of 25 microM, at which this agent selectively inhibits calmodulin-dependent protein kinase. These results suggest that PKC activation plays an important role in desmoplakin translocation from the cytoplasm to the plasma membrane as one of the processes of calcium-induced desmosome formation.     

Effects of A23187 upon cortical granule exocytosis in eggs of Brachydanio

Summary The effects of the divalent ionophore A23187 upon unfertilized eggs of the freshwater teleost fish, Brachydanio rerio, have been examined by light, scanning (SEM) and transmission (TEM) electron microscopy. Treatment of eggs with micromolar amounts (1 M, 10 M) of A23187 triggers cortical granule exocytosis and elevation of the chorion. However, the exocytosis of cortical granules in ionophore-activated eggs is explosive and occurs more rapidly than in eggs naturally activated in conditioned tap water. Eggs treated with A23187 in a medium lacking extra-cellular calcium also show cortical granule exocytosis, suggesting strongly that egg activation in Brachydanio results from release of calcium primarily from intracellular stores; however, there is a distinct delay in the onset of cortical granule breakdown. Unfertilized eggs exposed to A23187 for 1–5 min show noticeable disturbances in cell surface topography, including loss of microplicae and the appearance of prominent membrane-limited blebs.To determine if cortical granule exocytosis is self-propagating once initiated, A23187 was applied to a localized portion of the unfertilized egg surface, using either a G-50 sephadex gel bead or a 1 mm glass capillary tube. Eggs placed in continuous contact for 15 min with a bead coated with 10 M A23187 show neither exocytosis of cortical granules nor elevation of the chorion. All eggs exhibit exocytosis when positioned against a glass rod coated with 1 M A23187. The cortical granule breakdown is partial and restricted to less than 50% of the egg surface in most cells. The complete exocytosis of cortical granules in the zebra danio egg appears to require the stimulation and release of calcium from multiple sites over the cortex.     

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.     

Platelet-activating factor phosphatidate,but not platelet-activating factor,is a powerful calcium ionophore in the human red cell

Platelet-activating factor (1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, PAF) is a potent inducer of shape-change, aggregation and secretion in platelets. PAF causes a rapid increase in intracellular calcium, but has no calcium gating effect in intact lipid bilayers. Human red cells (RBC) did not metabolize either PAF or PAF-phosphatidate (PAF-PA). While PAF (10 μM) was devoid of calcium ionophoretic activity, PAF-PA (1–5 μM) stimulated calcium influx into intact human RBC. In addition, PAF-PA (1–10 μM), but not PAF (10 μM), elicited a series of satellite effects related to the rise of intracellular calcium: 1) increased efflux of intracellular potassium (Gàrdos effect); 2) alkalinization of unbuffered RBC suspensions; 3) stimulation of ATP consumption and production, and enhancement of glycolytic flux with crossover at the glyceraldehyde 3-phosphate dehydrogenase step. These effects exactly duplicate those brought about by the calcium ionophore A23187. The ionophoretic potency of PAF-PA was about half that of A23187. Approximately the same concentrations of PAF-PA as those that stimulate calcium influx into RBC elicit full aggregatory response in human platelets. It is possible that transformation of PAF into PAF-PA by the combined action of phospholipase C and diacylglycerol kinase contributes to the increase of calcium influx in platelets.     

Physiological responses of sea urchin eggs to stimulation by calcium ionophore A23187 analysed with protease inhibitors

Protease inhibitors were used to study certain physiological responses (secretion of the cortical granule protease, altered resceptively to sperm penetration, initiation of cell division and embryogenesis) of sea urchin eggs to stimulation by calcium ionophore A23187. Protease activity in the secretory product released from the eggs 5 min after insemination or parthenogenetic activation with ionophore was completely inhibited by soybean trypsin inhibitor (SBTI), antipain (Ap), and leupeptin (Lp). A barrier was established to prevent subsequently added sperm from penetrating (fertilizing) ionophore-activated eggs, co-incident with the elevation of the fertilization membrane. These processes were retarded by inhibitors of the cortical granule protease in ionophore-activated eggs, just as they are when eggs are initially stimulated by sperm at fertilization. A23187-activated eggs did not divide unless they had been secondarily fertilized by sperm, even if the ionophore was subsequently removed by extensive washing. However, ionophore-activated eggs that were penetrated by a single spermatozoan in SBTI developed into normal larvae under similar conditions. These results suggest that A23187 may be an incomplete parthenogenetic agent because it cannot stimulate eggs to assemble centrioles required to organize the mitotic apparatus. The centrioles are normally provided by the sperm during fertilization. A23187 may also be toxic to the eggs. Furthermore, since cortical granules are secretory organelles, the data suggest a possible functional relationship between calcium ions and protease activation in stimulus-secretion coupling in sea urchin eggs at fertilization.     

Release of mouse eggs from metaphase arrest by protein synthesis inhibition in the absence of a calcium signal or microtubule assembly

Mouse egg activation, which includes release from meiotic metaphase II arrest, results from fertilization-induced increase in intracellular calcium concentration ([Ca²⁺]_i). However, during egg activation caused by exposure to the protein synthesis inhibitor, cycloheximide, [Ca²⁺]_i did not change. Although eggs fertilized in the presence of microtubule inhibitors remain arrested at metaphase, eggs treated for 32 hr with cycloheximide and the microtubule inhibitor, colcemid, formed nuclei. In untreated eggs aged in culture for 24 hr, the microtubule spindles became deformed. These eggs formed nuclei after exposure to cycloheximide, but not the calcium ionophore A23187. Our results indicate that eggs in which protein synthesis is inhibited are released from metaphase without an increase in [Ca²⁺]_i, and despite disruption of the Spindle. © 1995 Wiley-Liss, Inc.