Activation Of Xenopus Eggs Research Articles

Evidence that phosphatidylinositol 3-kinase is involved in sperm-induced tyrosine kinase signaling in Xenopus egg fertilization

BackgroundStudies have examined the function of PI 3-kinase in the early developmental processes that operate in oocytes or early embryos of various species. However, the roles of egg-associated PI 3-kinase and Akt, especially in signal transduction at fertilization, are not well understood.ResultsHere we show that in Xenopus eggs, a potent inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), LY294002 inhibits sperm-induced activation of the tyrosine kinase Src and a transient increase in the intracellular concentration of Ca2+ at fertilization. LY294002 also inhibits sperm-induced dephosphorylation of mitogen-activated protein kinase, breakdown of cyclin B2 and Mos, and first embryonic cleavage, all of which are events of Ca2+-dependent egg activation. In fertilized eggs, an 85-kDa subunit of PI 3-kinase (p85) undergoes a transient translocation to the low-density, detergent-insoluble membranes (membrane microdomains) where Src tyrosine kinase signaling is operating. However, the tyrosine phosphorylation of p85 in fertilized eggs is not as evident as that in H2O2-activated eggs, arguing against the possibility that PI 3-kinase is activated by Src phosphorylation. Nevertheless, sperm-induced activation of PI 3-kinase has been demonstrated by the finding that Akt, a serine/threonine-specific protein kinase, is phosphorylated at threonine-308. The threonine-phosphorylated Akt also localizes to the membrane microdomains of fertilized eggs. Application of bp(V), an inhibitor of PTEN that dephosphorylates PIP3, the enzymatic product of PI 3-kinase, promotes parthenogenetic activation of Xenopus eggs. In vitro kinase assays demonstrate that PIP3 activates Src in a dose-dependent manner.ConclusionsThese results suggest that PI 3-kinase is involved in sperm-induced egg activation via production of PIP3 that would act as a positive regulator of the Src signaling pathway in Xenopus fertilization.

Uroplakin III, a novel Src substrate in Xenopus egg rafts, is a target for sperm protease essential for fertilization

In a previous study, we identified Xenopus egg uroplakin III (xUPIII), a single-transmembrane protein that localized to lipid/membrane rafts and was tyrosine-phosphorylated upon fertilization. An antibody against the xUPIII extracellular domain abolishes fertilization, suggesting that xUPIII acts not only as tyrosine kinase substrate but also as a receptor for sperm. Previously, it has been shown that the protease cathepsin B can promote a transient Ca 2+ release and egg activation as seen in fertilized eggs (Mizote, A., Okamoto, S., Iwao, Y., 1999. Activation of Xenopus eggs by proteases: possible involvement of a sperm protease in fertilization. Dev. Biol. 208, 79–92). Here, we show that activation of Xenopus eggs by cathepsin B is accompanied by tyrosine phosphorylation of egg-raft-associated Src, phospholipase Cγ, and xUPIII. Cathepsin B also promotes a partial digestion of xUPIII both in vitro and in vivo. A synthetic xUPIII-GRR peptide, which contains a potential proteolytic site, inhibits the cathepsin-B-mediated proteolysis and tyrosine phosphorylation of xUPIII and egg activation. Importantly, this peptide also inhibits sperm-induced tyrosine phosphorylation of xUPIII and egg activation. Protease activity that digests xUPIII in an xUPIII-GRR peptide-sensitive manner is present in Xenopus sperm. Several protease inhibitors, which have been identified to be inhibitory toward Xenopus fertilization, are shown to inhibit sperm-induced tyrosine phosphorylation of xUPIII. Uroplakin Ib, a tetraspanin UP member, is found to be associated with xUPIII in egg rafts. Our results highlight novel mechanisms of fertilization signaling by which xUPIII serves as a potential target for sperm protease essential for fertilization.

A PKC wave follows the calcium wave after activation of Xenopus eggs

Calcium waves are well-known hallmarks of egg activation that trigger resumption of the cell cycle and development of the embryo. These waves rapidly and efficiently assure that activation signals are transmitted to all regions of the egg. Although the mechanism by which the calcium wave propagates across an egg as large as that of Xenopus is not known, two models prevail. One model is a wave of calcium-induced calcium release (CICR) and the other is propagation by inositol-induced calcium release (IICR). IICR requires a wave of phosphatidylinositol 4,5-bisphosphate (PIP 2) hydrolysis, generating two second messengers, IP 3, which then releases calcium and DAG, which activates protein kinase C (PKC). We show here that a wave of PKC-green fluorescent protein travels across the egg immediately following, and at the same velocity as, the calcium wave. This is the first example of a PKC wave in a vertebrate egg and supports the IICR model of wave propagation.

Hydrogen peroxide induces Src family tyrosine kinase-dependent activation of Xenopus eggs.

Fertilization is accompanied by a rapid and transient calcium release in eggs, which is required for the onset of zygotic developmental program or 'egg activation'. Recently, it was found that Src family tyrosine kinase (SFK)-dependent phospholipase C (PLC) activity is necessary for the calcium transience in fertilized Xenopus eggs. The present study demonstrates that hydrogen peroxide (H2O2) stimulates protein-tyrosine phosphorylation in Xenopus eggs, which occurs primarily in the egg cortex of the animal hemisphere as revealed by indirect immunofluorescence study. Egg SFK was found to be upregulated by H2O2 while the SFK-specific inhibitor PP1 effectively blocked H2O2-induced tyrosine phosphorylation. As in fertilized eggs, PLCgamma, but not Shc, was tyrosine-phosphorylated in H2O2-treated eggs. H2O2 also caused inositol 1,4,5-trisphosphate (IP3) production and sustained calcium release. After limited application of H2O2, elevated SFK activity and tyrosine phosphorylation were quickly reversed. Under such conditions, eggs showed cortical contraction and dephosphorylation of p42 MAP kinase, both of which are indicative of egg activation. These egg activation events, as well as H2O2-induced IP3 production and calcium release, were sensitive to PP1 and PLC inhibitor U-73122. Together, the present study demonstrated that H2O2 can mimic, at least in part, early events of Xenopus egg activation that require an SFK-dependent PLC pathway.

Activation of Xenopus Eggs by the Kinase Inhibitor 6-DMAP Suggests a Differential Regulation of Cyclin B and p39mos 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 p39mos and MAPK play a part in the cytostatic activity whereas p34cdc2 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 p39mos 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 p39mos.

Activation ofXenopusEggs by Proteases: Possible Involvement of a Sperm Protease in Fertilization

Egg activation in cross-fertilization betweenXenopuseggs andCynopssperm may be caused by a protease activity against Boc-Gly-Arg-Arg-MCA in the sperm acrosome. To determine the role of the sperm protease in fertilization, the protease was purified fromCynopssperm using several chromatographic techniques. We found that purified sperm protease readily hydrolyzes Boc-Gly-Arg-Arg-MCA and Z-Arg-Arg-MCA, that protease activity was inhibited by the trypsin inhibitors aprotinin and leupeptin, and that not only the purified protease, but also cathepsin B, induces activation inXenopuseggs. We inseminated unfertilizedXenopuseggs with homologous sperm in the presence of various peptidyl MCA substrates or protease inhibitors and demonstrated that trypsin inhibitors or MCA substrates containing Arg-Arg-MCA reversibly inhibited fertilization of both fully jellied and denuded eggs. Sperm motility was not affected by the reagents. An extract obtained fromXenopussperm showed hydrolytic activity against Boc-Gly-Arg-Arg-MCA, Z-Arg-Arg-MCA, and Arg-MCA. These results suggest that the tryptic protease inXenopussperm is involved in fertilization, most likely by participating in egg activation.

Involvement of protein-tyrosine phosphorylation and dephosphorylation in sperm-induced Xenopus egg activation

We have analyzed tyrosine-phosphorylated proteins in Xenopus laevis eggs before and after fertilization by immunoblotting with anti-phosphotyrosine antibody. A number of egg proteins with different subcellular distribution became tyrosine-phosphorylated or dephosphorylated within 30 min after insemination. Tyrosine kinase-specific inhibitors genistein and herbimycin A were found to inhibit sperm-induced egg activation judged by the egg cortical contraction. Surprisingly, sodium orthovanadate, a tyrosine phosphatase inhibitor, also inhibited the egg activation. Moreover, we found that fertilization-dependent tyrosine dephosphorylation of 42-kDa mitogen-activated protein kinase was inhibited in genistein-treated eggs. These results suggest that both protein-tyrosine phosphorylation and dephosphorylation pathways play an important role in the sperm-induced Xenopus egg activation.

Activation ofXenopusEggs by RGD-Containing Peptides Accompanied by Intracellular Ca2+Release

To investigate the mechanism of egg activation inXenopus,unfertilized eggs were treated with various oligopeptides related to the RGD sequence-containing peptides. When dejellied eggs were treated with RGDS at concentrations higher than 250 μM,the eggs produced a positive-going activation potential concomitant with intracellular Ca2+release. The eggs underwent cortical granule breakdown, cortical contraction, and resumption of meiosis. The activation by RGDS was independent of the voltage of egg membranes. GRGD, GRGDTP, and RGPFPI caused activation in some eggs, but RGES, RFDS, GPRP amide, and GPA did not. Denuded eggs were activated by Sepharose beads bound to RGDS, indicating an interaction of the RGD sequence with receptor molecules on the egg plasma membrane. The activation by RGDS did not occur under a Ca2+- and Mg2+-free condition, but occurred in a Ca2+-free solution containing Mg2+, concomitant with an intracellular Ca2+release. These results indicate that a receptor of the RGD sequence on egg plasma membrane causes egg activation through an intracellular signal transduction system. A sperm agonist including the RGD sequence may play an important role in egg activation through the egg membrane receptor during the normal fertilization process.

Activation of the Proteasome duringXenopusEgg Activation Implies a Link between Proteasome Activation and Intracellular Calcium Release

Ubiquitin-dependent proteolysis is required for cell cycle progression. Here, we demonstrate that the proteasome is activated duringin vivo Xenopusegg activation, induced by treatment with the calcium ionophore A23187. It was found that activation is due to the calcium-induced assembly of the 26 S proteasome from the 20 S proteasome. We propose that proteasome activation is regulated by cell cycle calcium transients, which are controlled upstream by an endogenous cell cycle oscillator that is independent of the cyclin-dependent kinase cycle.

Activation of Xenopus Eggs by Cynops Sperm Extract is Dependent upon Both Extra-and Intra-Cellular Ca Activities

Abstract When unfertilized Xenopus eggs were treated by Cynops sperm extract in 10% Steinberg's solution (SB), egg's membranes hyperpolarized to about −37 mV and then depolarized to elicit a positive-going potential amounting to about +34 mV. The eggs underwent cortical contraction and resumption of meiosis. Activation of eggs in various external solutions indicates that the hyperpolarization is due mainly to opening of Na channels, but the positive-going potential is due to Cl channels on the egg's plasma membranes. Since the activation was inhibited by CdCl2, CoCl2, or NiCl2 as well as by amiloride, Ca influx through Ca channels is necessary for the activation by the sperm extract. A propagative intracellular Ca release was induced not only by Cynops sperm, but also by their sperm extract. Injection of BAPTA or heparin into the eggs completely inhibited activation, indicating that egg activation requires an intracellular Ca release dependent upon receptors for inositol 1,4,5-trisphosphate.

Activation of Xenopus Eggs by an Extract of Cynops Sperm: (fertilization/egg-activation/polyspermy block/sperm protease).

An extract obtained from Cynops sperm induced the activation of both Cynops and Xenopus eggs with accompanying changes in the potential of the egg membrane that were quite similar to those caused by the Cynops sperm. The activation-inducing properties of the extract were abolished by treatment with proteinase K or by heating (60°C, 15 min) and were associated with a protease activity against peptidyl Arg-MCA substrates. The activation of Xenopus eggs by the extract was inhibited by those substrates, or by protease inhibitors, aprotinin or leupeptin. The protease activity was localized in the acrosomal region of Cynops sperm. The activation of Xenopus eggs by the extract was prevented when the exterior concentration of Ca2+ ions, [Ca2+ ]0 , was reduced to 1.5 μM, but it was enhanced when [Ca2+ ]0 was increased to 340 μM. The activation of Xenopus eggs by the extract was not affected by positive clamping when [Ca2+ ]0 was 340 μM. These results suggest that the sperm extract contains a protease that causes an increase in the influx of Ca2+ ions that results in voltage-insensitive activation of the egg.

Degradation of Mos by the N-terminal proline (Pro2)-dependent ubiquitin pathway on fertilization of Xenopus eggs: possible significance of natural selection for Pro2 in Mos.

The c-mos proto-oncogene product (Mos), an essential component of the cytostatic factor responsible for meiotic arrest in vertebrate eggs, undergoes specific proteolysis soon after fertilization or activation of Xenopus eggs. To determine the degradation pathway of Mos on egg activation, various Mos mutants were expressed in Xenopus eggs and their degradation on egg activation was examined. Mos degradation absolutely required its penultimate proline (Pro2) residue and dephosphorylation of the adjacent serine (Ser3) residue. These degradation signals were essentially the same as those of Mos in meiosis I of Xenopus oocyte maturation, where Mos has been shown to be degraded by the 'second-codon rule'-based ubiquitin pathway. To test whether Mos degradation on egg activation is also mediated by the ubiquitin pathway, we attempted to identify and abrogate a specific ubiquitination site(s) in Mos. We show that the major ubiquitination site in Mos is a Lys34 residue and that replacement of this residue with a non-ubiquitinatable Arg residue markedly enhances the stability of Mos on egg activation. These results indicate that the degradation of Mos on egg activation or fertilization is mediated primarily by the N-terminal Pro2-dependent ubiquitin pathway, as in meiosis I of oocyte maturation. The N-terminal Pro2 residue of Mos appears to be naturally selected primarily for its degradation on fertilization, rather than that in meiosis I.

Activation of Xenopus laevis eggs in the absence of intracellular Ca activity by the protein phosphorylation inhibitor, 6-dimethylaminopurine (6-DMAP).

Xenopus laevis eggs pricked or microinjected with water or saline in medium containing a limited quantity of free Ca (1.0 to 2.0 microM) remain unactivated for at least 6 hr, even after transfer to oocyte medium containing Ca at higher concentrations (0.5-1.0 mM). These injected eggs, when later pricked in oocyte medium or exposed to A23187 or urethane are fully capable of activation. This confirms the observations of Wangh ('89). However, eggs injected in this Ca-limited medium (CaLM) with 6-DMAP as well as those simply exposed to this drug undergo changes characteristic of activation, including cortical contraction, cortical granule breakdown, a loss of MPF and CSF activities, and pronuclear formation. The time required for 6-DMAP to induce egg activation is inversely correlated to its concentration. Interestingly, eggs that have been injected with EGTA, and thus are unable to respond to activation stimuli such as pricking and A23187 or urethane treatment, can also be activated by exposure to 6-DMAP. In contrast, eggs exposed to or injected with a 6-DMAP analogue (6-aminopurine or puromycin) or a protein synthesis inhibitor (cycloheximide or emetine or puromycin) are not activated. As well, eggs injected in CaLM with 6-DMAP simultaneously with a phosphatase inhibitor (NaF or ammonium molybdate) fail to become activated. Although 6-DMAP-activated eggs remain at the pronucleus stage so long as 6-DMAP is present, they resume cell cycle activities after the drug is withdrawn. They form cleavage furrows, disassemble pronuclear envelopes, and recondense chromosomes. Also, MPF activity reappears and cycles at least twice, peaking each time shortly before cleavage furrow formation. These results suggest that activation of Xenopus eggs arrested at metaphase II by inhibition of protein phosphorylation does not require intracellular Ca release and that maintenance of the egg at metaphase II depends upon continuous protein phosphorylation.

Independent inactivation of MPF and cytostatic factor (Mos) upon fertilization of Xenopus eggs

In vertebrates, mature eggs are arrested at the second meiotic metaphase by the cytostatic factor (CSF), now known to be the c-mos proto-oncogene product (Mos). Fertilization or egg activation triggers a transient increase in the cytoplasmic free calcium and releases the meiotic arrest by inactivating maturation/mitosis-promoting factor (MPF). CSF or Mos, which is also inactivated by the calcium transient, seems to stabilize MPF in mature eggs and CSF-injected embryos. Thus, it was assumed that CSF inactivation is the primary cause of MPF inactivation on meiotic release. We have directly compared the degradation kinetics of CSF (Mos) and MPF during meiotic release, using the same batch of Xenopus eggs. We report here that, at the molecular level, cyclin subunits of MPF are degraded before Mos is degraded and, at the physiological level, that MPF activity is inactivated before CSF activity during activation of Xenopus eggs. These results, in conjunction with circumstantial evidence, support the novel view that a calcium transient on fertilization induces a CSF-independent pathway for MPF inactivation, whereas CSF inactivation during meiotic release serves only to allow the fertilized egg to enter mitosis.

Parallel pathways of cell cycle control during Xenopus egg activation.

Transit from M phase into interphase in many eukaryotic cells is preceded by an increase in intracellular free calcium ([Ca2+]i), which may act via calcium-dependent enzymes to trigger the M-phase/interphase transition. To test the role of the calcium- and phospholipid-dependent enzyme protein kinase C (PKC) in the M-phase/interphase transition, PKC was activated in M-phase-arrested Xenopus eggs by treatment with the phorbol ester phorbol 12-myristate 13-acetate under conditions that prevent a rise in [Ca2+]i and activation of other calcium-dependent enzymes. Under these conditions, several cellular events characteristic of transit into interphase occur: sperm chromatin decondenses, the Golgi and the nuclear envelope reassemble, and endocytosis resumes. These events are also triggered by treatment of eggs with the diacylglycerol 1,2-dioctanoyl-sn-glycerol. Surprisingly, the activity of M-phase-promoting factor (MPF), a universal regulator of M phase, remains high under these conditions. If [Ca2+]i is subsequently raised, MPF activity is rapidly destroyed. Similarly, lysates made from eggs treated with phorbol 12-myristate 13-acetate support sperm chromatin decondensation in vitro and yet retain high MPF activity, measured either as the ability to induce meiotic resumption in oocytes or as histone H1 kinase activity. These effects are not triggered by the 4 alpha-phorbol ester isomer, which does not activate PKC, and are sensitive to the PKC "pseudosubstrate" peptide. The results suggest that two, parallel signals are generated by the rise in [Ca2+]i both of which contribute to cell cycle regulation. One pathway inactivates MPF; the other pathway activates PKC.

Intracellular pH and the increase in protein synthesis accompanying activation of Xenopus eggs

Metabolic activation following egg fertilization corresponds to an increase in protein synthesis and the initiation of DNA synthesis, which lead to cell division and development of the embryo. Since in several biological systems protein synthesis is regulated by intracellular pH (pHi), we have decided to investigate the situation during Xenopus egg activation. We confirmed that egg activation is accompanied by a pHi rise of 0.3 pH unit. Measurements of the rates of protein synthesis is unactivated and activated eggs, after microinjection of 3H-leucine, demonstrated that activation was followed by a 2.5-fold increase. Treatment of unactivated eggs with weak bases also increased pHi, but did not result in an increase in the rate of protein synthesis. Moreover, in vitro translation in cytoplasmic extracts was found to be pH-independent, at least between 6.8 and 8.2.

Intracellular pH and the increase in protein synthesis accompanying activation of Xenopus eggs

Metabolic activation following egg fertilization corresponds to an increase in protein synthesis and the initiation of DNA synthesis, which lead to cell division and development of the embryo. Since in several biological systems protein synthesis is regulated by intracellular pH (pHi), we have decided to investigate the situation during Xenopus egg activation. We confirmed that egg activation is accompanied by a pHi rise of 0.3 pH unit. Measurements of the rates of protein synthesis in unactivated and activated eggs, after microinjection of 3H-leucine, demonstrated that activation was followed by a 2.5-fold increase. Treatment of unactivated eggs with weak bases also increased pHi, but did not result in an increase in the rate of protein synthesis. Moreover, in vitro translation in cytoplasmic extracts was found to be pH-independent, at least between 6.8 and 8.2.

External Na + inhibits Ca 2+-ionophore activation of Xenopus eggs

In medium containing 8.25 m M NaCl, eggs of Xenopus laevis can be activated by threshold concentrations (3 to 5 × 10 −8 M) of the divalent cation ionophore, A23187. Activation by threshold concentrations of A23187 is reduced substantially when the concentration of NaCl in the medium is raised to 40 m M. Ion substitution experiments with NaI, Na isethionate, and choline chloride demonstrate that the inhibitory effect is due to Na + rather than Cl −. The inhibitory effect of 40 m M Na + is blocked by the sodium influx inhibitor, amiloride (1 m M), and by 1 m M verapamil and 1 m M La 3+. Elevation of intracellular pH (pH i) with NH 4Cl markedly increased the effectiveness of threshold levels of A23187, as evidenced by hypercontraction of the cortex. Neither amiloride nor changes in extracellular Na + concentration alter pH i, however. Changing the concentration of extracellular Ca 2+ had no effect on activation by A23187, regardless of the concentration of Na + in the extracellular medium. The effect of Na + on ionophore-induced activation is discussed in terms of alternative hypotheses, including a sodium-calcium exchange mechanism that operates in somatic cells to maintain low intracellular concentrations of Ca 2+.