Macrophage migration inhibitory factor and DJ-1 in gastric cancer: differences between high-incidence and low-incidence areas.

BACKGROUND: There is a need for sensitive and specific blood-borne markers for the detection of gastric cancer. Raised serum macrophage inhibitory factor (MIF) levels have been proposed as a marker for gastric cancer diagnosis but, to date, studies have only encompassed patients from high-incidence areas. METHODS: We have compared the serum concentration of MIF in a large cohort of UK and Japanese gastric cancer patients, together with appropriate control subjects (age and gender matched). Carcinoembryonic antigen and H. pylori IgG were also measured, as was DJ-1, a novel candidate protein biomarker identified by analysis of gastric cancer cell line secretomes. RESULTS: Marked elevations of the serum concentration of MIF and DJ-1 were seen in Japanese patients with gastric cancer compared with Japanese controls, a trend not seen in the UK cohort. These results could not be accounted for by differences in age, disease stage or H. pylori status. CONCLUSION: In regions of high, but not low incidence of gastric cancer, both MIF and DJ-1 have elevated serum concentrations in gastric cancer patients, compared with controls. This suggests that differing mechanisms of disease pathogenesis may be at play in high- and low-incidence regions.

Identification of macrophage migration inhibitory factor and human neutrophil peptides 1-3 as potential biomarkers for gastric cancer.

BACKGROUND: Proteomic methods have the potential to meet the urgent need for better cancer biomarkers. We have used a range of proteomic analyses of serum and tissue from gastric cancer patients and relevant controls to discover biomarkers for gastric cancer. METHODS: Surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI) and antibody arrays were used to compare protein expression in 21 pairs of gastric cancer tissue and adjacent normal mucosa and serum from 51 gastric cancer patients and 29 patients with benign gastric diseases. Expression differences were confirmed by enzyme-linked immunosorbent assay. RESULTS: Tissue analysis shows human neutrophil peptides 1-3 (HNPs 1-3) elevated 10-fold (P=0.001) in gastric cancer relative to adjacent normal mucosa. Macrophage migration inhibitory factor (MIF) was increased five-fold (P=1.84 x 10(-7)) in the serum of gastric cancer patients relative to individuals with benign gastric disease. The large increase in MIF concentration in serum gives an area under the receiver operating characteristic curve of 0.85. CONCLUSIONS: Proteomic analyses of serum and tissue indicate that HNPs 1-3 and MIF have potential as biomarkers for gastric cancer. In particular MIF may be useful, either alone or in combination with other markers, for diagnosing and monitoring gastric cancer.

Reconstitution of the L-leucine-H+ cotransporter of the plasma membrane from Chang liver cells into proteoliposomes.

L-Leucine is cotransported with H+ in the plasma membrane of Chang liver cells (Mitsumoto, Y. et al. (1986) J. Biol. Chem. 261, 4549). The leucine transport system was solubilized from the plasma membrane of the cells with ocytl glucoside and reconstituted in proteoliposomes prepared by a rapid dilution of a mixture of the solubilized proteins, octyl glucoside and liposomes. The proteoliposomes exhibited H(+)-gradient and electrical potential-stimulated leucine uptake. The H(+)-gradient-stimulated leucine uptake could be completely inhibited by carbonyl cyanide p-trifluoro-methoxyphenylhydrazone (FCCP) and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH). The stimulatory effect of H+ gradient on leucine uptake was shown to be mainly due to decrease of the Km, but not to change of the Vmax, of the transport kinetics. These results suggest that the leucine-H+ cotransporter is solubilized and reconstituted into proteoliposomes.

Effect of N-ethylmaleimide on leucine transport in the Chang liver cell. II. Effect on the kinetics of Na+-independent transport.

The Na+-independent leucine transport system is resolved into two components by their different affinity (Km about 44 microM and 8.0 mM) for leucine in the Chang liver cell. Treatment of the cells with N-ethylmaleimide (1 mM) specifically stimulates the high-affinity component of the Na+-independent system by greatly increasing its Vmax value, whereas the Vmax value of the low-affinity component is markedly lowered. The stimulatory effect of N-ethylmaleimide on leucine transport is reduced by prior treatment of the cells with 2,4-dinitrophenol, but this phenomenon seems to be irrelevant to the ATP-depleting action of the uncoupler. The treatment with 2,4-dinitrophenol has been found not to be inhibitory on the subsequent Na+-independent leucine uptake itself. Treatment with dibucaine, a phospholipid-interacting drug, also reduces to varying degrees (depending on its concentration) the stimulatory effect of N-ethylmaleimide on the subsequent leucine uptake, although pretreatment with dibucaine can stimulate the Na+-independent leucine uptake itself. We conclude that the stimulatory effect of N-ethylmaleimide on leucine transport is not correlated with the energy level of cell, but involves the perturbation of the membrane bilayer structures.

Effect of ionic strength on the membrane fluidity of rabbit intestinal brush-border membranes. A fluorescence probe study.

The effect of ionic strength on the fluidity of rabbit intestinal brush-border membranes has been studied using two fluorescence probes, pyrene and 1-anilino-8-naphthalene sulfonate (ANS). The imposition of a potential gradient on the pyrene-probed membrane vesicles (out greater than in) with increasing NaCl concentration in the medium resulted in a marked enhancement of the excimer formation efficiency, accompanied by a decrease in the ratio of fluorescence intensities of the probe at 392 and 375 nm. Fluorescence polarization of the pyrene-membrane complex is independent of temperature in the absence of salts, while it is dependent on temperature from 10 to 47 degrees C in the presence of salts, as shown by the thermal Perrin plots of polarization. It has been demonstrated that there is a linear relationship between the changes in the pyrene excimer formation efficiency in the membranes and of the values of the binding parameters of ANS for the membranes. From these results, it is suggested that the lipid phase of the membranes becomes more fluid by shielding negatively charged groups of the membrane surface and that there is a fairly close correlation between the membrane organization and the membrane surface charge density.

Effect of N-ethylmaleimide on leucine transport in Chang liver cells. I. stimulatory effect on Na+ -independent transport at low concentrations of leucine.

Pretreatment of Chang liver cells with N-ethylmaleimide (0.5 or 1 mM) stimulated Na+ -independent uptake of leucine at low concentrations (less than or equal to 1 mM). The stimulatory effect of N-ethylmaleimide on the uptake of leucine measured in Na+ -replete medium was completely blocked by the addition of beta-2-aminobicyclo[2,2,1]heptane-2-carboxylate (5 mM), which shows that the L system participates in the stimulation. The Na+ -dependent uptake of glycine was depressed by N-ethylmaleimide pretreatment. The stimulation of the Na+ -independent component of leucine uptake continued for at least 30 min after N-ethylmaleimide treatment, while the inhibition of glycine uptake was progressive with time and the Na+ -dependent uptake of leucine became depressed later, after the treatment. It has been demonstrated that treatment of cells with N-ethylmaleimide is capable of increasing the Na+ -independent influx of leucine and at the same time slightly decreasing the efflux of it. These results suggest that N-ethylmaleimide attacks the Na+ -independent system of amino acid transport at the reactive SH groups(s) of relevant protein(s) in favor of specific activation of that system in this cell.

Leucine transport in relation to the activities of Na+-H+ antiporter and Na+/K+ pump stimulated by serum and a tumor promoter.

Fetal calf serum and 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the rate of leucine uptake by Chang liver cells in Na+-containing medium. Addition of monensin to the incubation medium also increased the leucine uptake. All these agents were capable of raising the cytoplasmic pH, which was blocked by a prior addition of amiloride or removing Na+ from assay medium, suggesting activation of Na+-H+ exchange across the cell membrane by fetal calf serum and TPA. The stimulation of leucine uptake by monensin and fetal calf serum was blocked completely or incompletely by addition of ouabain or amiloride. The basal and fetal-calf-serum- or TPA-stimulated leucine uptake was extensively depressed by the presence of an excess of 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid in the incubation medium. Based on these results it is proposed that the transport of leucine by the system L is stimulated by fetal calf serum and TPA with a high concentration of Na+ outside the cells as a result of alkalinization of the cytoplasm and coordinated activation of (Na+ + K+)-ATPase by these stimulators to maintain the transmembrane Na+ gradient and also hyperpolarize the cell membrane.

Effect of neuraminidase treatment on the lipid fluidity of the intestinal brush-border membranes.

The effect of neuraminidase treatment on the lipid fluidity of the porcine intestinal brush-border membranes was studied using two fluorescence dyes, pyrene and 1,6-diphenyl-1,3,5-hexatriene. By treatment of the membranes with neuraminidase, the fluorescence parameters of pyrene-labeled membranes changed; i.e., a shift of thermal transition temperature, an increase in the fluorescence quenching rate for Tl+ and a decrease in the fluorescence lifetime. These results suggest that the environmental properties around the dye molecules in the membranes change sensitively upon neuraminidase treatment. Perturbation of the lipid domain in the membranes associated with neuraminidase treatment is also demonstrated by a stimulated solubilization of diphenylhexatriene molecules in the membrane lipids, an increased quenching efficiency with Tl+ and a decreased rotational correlation time of diphenylhexatriene-labeled membranes. Based on these results, we conclude that the lipid organization of the membranes is susceptible to neuraminidase treatment and that the membrane lipid fluidity increases by desialylation by the enzyme treatment.

Increase of the molecular rigidity of the protein conformation in the intestinal brush-border membranes by lipid peroxidation.

The effect of lipid peroxidation on the protein conformation of the porcine intestinal brush-border membranes was studied using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM). By a kinetic analysis of the reaction of the membranes with DACM, it was shown that the reaction rate of the SH groups (SHf) of the membrane proteins, whose reaction with the dye is very fast, decreases in proportion to the extent of thiobarbituric acid-reactive substance formation. The difference in the rate of the reaction of the SHf groups for DACM between the control and peroxidized membranes completely disappeared after denaturation of the proteins by treatment with guanidine hydrochloride. The reaction of DACM with the SHf groups of the control membranes accelerated when the temperature was increased with an apparent transition temperature between 25 degrees C and 30 degrees C. On the other hand, no transition was observed in the peroxidized membranes over the temperature range 20-43 degrees C. These results suggest that the conformation around the SHf groups of the proteins in the peroxidized membranes is apparently different from that in the control membranes. A modification of the conformation around the SH groups in the membrane proteins associated with lipid peroxidation was further demonstrated by finding that the quenching efficiency of the fluorescence of the DACM-labeled membranes by Tl+ was markedly decreased after lipid peroxidation. Based on these results, changes in the protein conformation of the porcine intestinal brush-border membranes by lipid peroxidation are discussed.

Effects of alpha-tocopherol on the lipid peroxidation and fluidity of porcine intestinal brush-border membranes.

The effect of alpha-tocopherol on the lipid fluidity of porcine intestinal brush-border membranes was studied using pyrene as a fluorescent probe. Addition of alpha-tocopherol to the medium decreased fluorescence intensity and lifetime, but increased the fluorescence polarization of pyrene-labeled membranes. beta-, gamma-, and delta-Tocopherols gave no appreciable effect on the fluorescence intensity and polarization of the complex. The apparent dissociation constant (3.1 +/- 0.12 microM) of the interaction of alpha-tocopherol with the membranes, estimated from the change in the fluorescence intensity with varying concentrations of alpha-tocopherol, was in good agreement with the concentration required to cause the half-maximal inhibition of lipid peroxidation of the membranes performed by incubation with 100 microM ascorbic acid and 10 microM Fe2+. Decrease of the slope in the thermal Perrin plot of the polarization of pyrene-labeled membranes by alpha-tocopherol suggests that the movement of pyrene molecules in the membranes is restricted by binding of the tocopherol. This interpretation was confirmed by an increased harmonic mean of the rotational relaxation time of the dye molecules in the membranes from 10.9 +/- 0.16 to 18.5 +/- 0.51 microseconds after addition of 25 microM alpha-tocopherol to the medium. The perturbation of lipid phase in the membranes induced by alpha-tocopherol was also suggested from a decreased quenching rate constant of pyrene fluorescence in the membranes for Tl+. Based on these results, the effect of alpha-tocopherol on the lipid fluidity of the membranes is discussed.

Ca2+-dependent ATP hydrolysis of the porcine intestinal brush-border membranes.

The brush-border membrane from the porcine small intestine possesses Ca2+-dependent ATPase activity. The Ca2+ stimulation of ATP hydrolysis by the membranes is biphasic with a high affinity (Km = 0.38 microM) and a low affinity (Km = 98.3 microM). Treatment of the membrane vesicles with n-heptylthioglucoside did not cause further increase of the Ca2+-ATPase activity. Mg2+ also stimulates the ATP hydrolysis in the absence of Ca2+ but decreases the Ca2+-ATPase activities at 0.59 and 200 microM free Ca2+. The Ca2+-ATPase activities are not inhibited by addition of vanadate, ouabain, sodium azide and alkaline phosphatase inhibitors (theophylline and L-phenylalanine), irrespective of the Ca2+ concentrations in medium. A specific calmodulin-inhibitor W-7 (up to 30 microM) also did not influence on the Ca2+-ATPase activities at 0.59 and 200 microM free Ca2+. The Ca2+-ATPase activities at 0.59 and 200 microM free Ca2+ show no specificity for ATP. ADP, GTP and CTP could also be used as substrates. From these results, it is suggested that the porcine intestinal brush-border membrane possesses Mg2+-independent Ca2+-ATPase activity and that the Ca2+-ATPase activities with biphasic responses for Ca2+ stimulation observed in the present study reside on the same protein. The physiological functions of the Ca2+-ATPase in the membranes, however, remain unknown at present.

A change in the lipid fluidity of the porcine intestinal brush-border membranes by lipid peroxidation. Studies using pyrene and fluorescent stearic acid derivatives.

The effect of lipid peroxidation on the lipid fluidity of porcine intestinal brush-border membranes was examined by measuring the rotational mobility and the accessibility to fluorescence quenchers (CH3COOT1, CuSO4 and KI) of pyrene or n-(9-anthroyloxy)stearic acid (n = 2 or 12) in the membranes. The harmonic mean of the rotational relaxation times of pyrene increased and the rate constants, kq, of the quenching reaction of pyrene and 2-(9-anthroyloxy)stearic acid incorporated in the membrane lipids decreased upon lipid peroxidation, indicating reduction of the lipid fluidity of the membranes by lipid peroxidation. In addition, the kq value of the reaction of 2-(9-anthroyloxy)stearic acid in the membranes with Cu2+ decreased in proportion to the amount of the products of lipid peroxidation. On the other hand, the kq value of the reaction of 12-(9-anthroyloxy)stearic acid with Cu2+ or I- was unaffected by lipid peroxidation. Based on these results, a localized change in the lipid fluidity of the membranes in association with lipid peroxidation has been discussed.

Stimulation of leucine transport by a mitogen through intracellular Ca2+ increase in human peripheral lymphocytes.

The effect of concanavalin A and ionophore A23187 on leucine uptake by human peripheral lymphocytes has been examined. Preincubation of the cells with 32 micrograms/ml concanavalin A or 0.1 microM A23187 increased leucine uptake by 67% and 100%, respectively. Both concanavalin A and A23187 could, within 2 min, induce a more than 2-fold increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i). This increase by concanavalin A was completely blocked by the addition of 0.1 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8) to incubation medium; TMB-8 partially blocked the action of A23187. The stimulation of leucine uptake by concanavalin A and A23187 was strongly inhibited by the presence of TMB-8 in the medium, whereas the basal uptake was not affected by this intracellular Ca2+ antagonist. Amiloride did not inhibit the stimulation of leucine uptake by concanavalin A. The concanavalin A- and A23187-induced elevation of [Ca2+]i was accompanied by membrane hyperpolarization. Concanavalin A-stimulated leucine uptake was greatly inhibited by the presence of an excess of 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid. These results indicate that the increase in [Ca2+]i may function as a signal of the stimulation by mitogen of leucine uptake mediated by system L, finally inducing membrane hyperpolarization in human lymphocyte.

Leucine transport-induced activation of the Na+/H+ exchanger in human peripheral lymphocytes.

Adjustment of amino-acid-induced cytoplasmic pH decrease by the Na+/H+ exchange system in human lymphocytes has been studied using a fluorometric technique to monitor the intracellular pH change. When the interior of lymphocytes is acidified by addition of nigericin to medium, cytoplasmic pH is immediately corrected toward its resting value. This recovery of the cytoplasmic pH depends on extracellular Na+ and is inhibited by amiloride. A temporary (less than 2 min) decrease in the cytoplasmic pH, followed by a slow recovery phase, was observed in incubation with 1.0 mM leucine in Na+-containing medium. This leucine-dependent decrease of cytoplasmic pH persisted longer when amiloride was added to the medium. Cytoplasmic pH recovery from the leucine-induced acidification depends on external Na+ concentration. Amiloride-sensitive Na+/H+ exchanger was stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) in the lymphocytes and preincubation of the cells with TPA partially prevented the leucine-induced cytoplasmic acidification. We conclude that human peripheral lymphocytes are provided with an amino acid-H+ cotransport system, which is cooperatively coupled to the amiloride-sensitive Na+/H+ exchanger to correct the cytoplasmic pH anomaly.

Effect of lipid peroxidation on membrane-bound Ca2+-ATPase activity of the intestinal brush-border membranes.

We have studied lipid peroxidation and Ca2+-ATPase activity of the porcine intestinal brush-border membranes using a oxygen-radical-generating system consisting of dithiothreitol (DTT)/Fe2+ and tert-butyl hydroperoxide (t-BuOOH). The rates of lipid peroxidation were measured by formation of thiobarbituric acid-reactive substances (TBAR) and conjugated diene. Incubation of the membranes with DTT/Fe2+ in the absence and presence of t-BuOOH resulted in a slight (about 20%) and a marked (about 50%) inhibition of Ca2+-ATPase activity, respectively. The degree of inhibition was dependent on the hydroperoxide concentration. Addition of thiourea effectively protected Ca2+-ATPase activity but catalase and superoxide dismutase showed a slight and no effect on protection of the ATPase activity, respectively. Results of kinetic studies on the ATPase activity with varying ATP and Ca2+ concentrations revealed that the decrease in the enzyme activity by treatment with these oxidizing agents is mainly due to decrease of the Vmax value. Modification of SH groups in the membrane proteins by thiol group reagents such as N-ethylmaleimide, monoiodoacetate and monoiodacetamide did not induce the inhibition of Ca2+-ATPase activity. From these results, it is suggested that inhibition of the ATPase activity of the membranes by treatment with DTT/Fe2+ in the presence and absence of t-BuOOH is dependent on lipid peroxidation and that oxidative modification of SH groups may not be directly involved to the loss of the ATPase activity. In addition, results of the fluorescence anisotropy measurements of pyrene-labeled membranes suggested that change in the Ca2+-ATPase activity is partly related to a decrease in the membrane lipid fluidity.

Changes in the fluorescence parameters of bound N-(1-pyrene) maleimide by lipid peroxidation of intestinal brush-border membranes.

Using a fluorogenic thiol reagent, N-(1-pyrene)maleimide (NPM), we have examined of lipid peroxidation on the microenvironment around SH groups of the membrane proteins in porcine intestinal brush-border membrane vesicles. The lipid peroxidation of the membranes was performed with various concentrations of t-butylhydroperoxide (t-BuOOH) in the presence of 100 microM ascorbic acid and 10 microM Fe2+. Treatment of NPM-labeled membranes with these oxidizing agents resulted in a decrease of the fluorescence lifetime, suggesting modification of the environmental properties around the bound dye. Measurement of the steady-state fluorescence anisotropy of the labeled membranes indicated restriction of the motion of the bound dye by the lipid peroxidation membranes. This interpretation was further supported by an elevation of the transition temperature of the anisotropy, a decrease in the quenching rate constant of the fluorescence with acrylamide and a decrease in the SH reactivity of the membrane proteins for NPM by lipid peroxidation. Based on these results, the possibility of conformation changes in the vicinity of SH groups in the membrane proteins associated with lipid peroxidation has been discussed.

Characterization of interaction between Tb3+ and porcine intestinal brush-border membranes.

Effects of ionic strength and temperature on the interaction between Tb3+ and porcine intestinal brush-border membrane vesicles were studied. When Tb3+ was added to the vesicle suspension, Tb3+ fluorescence increased with increasing concentration of Tb3+, showing a saturation. The apparent dissociation constant of one of at least two components of this binding reaction was estimated to be about 12.5 microM at 25 degrees C, pH 7.4. But the affinity of Tb3+ for the membrane vesicles was variable with changes of ionic strength and temperature. The affinity was lowered by addition of KCl to medium and by increase of temperature above 30 degrees C. In addition, temperature-induced change in the affinity of Tb3+ for the membranes was reversible over a temperature range from 13 to 46 degrees C. Temperature-dependence profiles of the excimer formation efficiency of pyrene-labeled membranes and of the harmonic mean of the rotational relaxation times of pyrene molecules in the membranes revealed that the phase transition of the membrane lipids occurs at about 30 degrees C. Based on these results, characteristics of Tb3+ binding to the membranes are discussed in relation to the nature of lipid phase and surface charges of the membranes.

Temperature-induced transitions of porcine intestinal brush border membranes.

Thermotropic transitions of the membrane components in porcine intestinal brush border membranes were studied by means of fluorimetry using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM), and a lipophilic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). 1. The reactivity of the sulfhydryl groups of the membrane proteins with DACM was dependent on temperature, with a transition point at about 33 degrees C. A conspicuous transition was also observed in the relation between temperature and the fluorescence intensity of DACM-labeled membranes at 35 degrees C. 2. Temperature dependence profiles of the solubilization of DPH in the membranes and of the fluorescence polarization of DPH-membrane complex suggested that the phase transition of the lipid from gel to liquid-crystalline state occurs over a temperature range of 30 to 35 degrees C. 3. Efficient fluorescence energy transfer was observed from tryptophan residues of the membrane proteins to DPH located in the lipid phase of the membranes, and its efficiency was extremely enhanced, dependent on temperature, above 35 degrees C. The intensity of the tryptophan fluorescence of the membrane proteins decreased with increasing temperature and a discontinuity was observed at about 33 degrees C. Based on these results, it may be concluded that there are co-operative interactions between proteins and lipids in the membranes and that the temperature-induced conformational changes of the membrane proteins are closely related to the dynamics of the hydrocarbon cores of the lipid.

The effect of ionic strength on the lipid peroxidation of porcine intestinal brush-border membrane vesicles.

The effects of salt concentration gradient (inside to outside) on the lipid peroxidation of porcine intestinal brush-border membrane vesicles have been studied and several interesting features of the peroxidation have been elucidated. The addition of dithiothreitol and Fe2+ is far more effective in induction of the lipid peroxidation than any of the other metal ion species tested (Fe3+, Cu2+, Ni2+, Zn2+ and Cr3+). The peroxidation rate of the membrane vesicles induced by dithiothreitol plus Fe2+ was sensitive for the incubation temperature and was increased with increase of the temperature. Imposition of an inward salt concentration gradient on the membrane vesicles preloaded with 300 mM mannitol by addition of 100 mM chloride of K+, Na+, Li+, Rb+, NH4+ or choline to medium produces a very large reduction of the lipid peroxidation induced by dithiothreitol plus Fe2+. The membrane peroxidation is depressed more with the mannitol (300 mM)-preloaded vesicles than with the K2SO4 (100 mM)-preloaded vesicles when they are incubated in medium containing 20-100 mM of K2SO4. Addition of membrane-permeant anions such as SCN- and I-, but not addition of NO3-, to incubation medium has been found to decrease markedly the lipid peroxidation of the mannitol-preloaded vesicles. From these results it is suggested that the lipid peroxidation of the brush-border membranes by addition of dithiothreitol plus Fe2+ is sensitively changed with change in ionic strength.

Spatiotemporal analysis of [Ca2+]i rises in mouse eggs after intracytoplasmic sperm injection (ICSI).

Intracytoplasmic sperm injection (ICSI) into mammalian eggs induces repetitive rises in intracellular Ca2+ concentration ([Ca2+]i) which are the pivotal signal in fertilization. Spatiotemporal aspects of [Ca2+]i rises following ICSI into the periphery of mouse eggs were investigated with high-speed confocal microscopy. The first Ca2+ response was generated 25-30 min after ICSI, when [Ca2+]i increased slowly and reached a certain level. The [Ca2+]i rise occurred synchronously over the ooplasm, attained the peak in 40-70 s, and lasted for 5-7 min. Succeeding Ca2+ responses occurred at intervals of 20-30 min, associated with the faster rate of [Ca2+]i rise and the shorter duration as Ca2+ oscillations progressed. The [Ca2+]i rises took the form of a wave that started from an arbitrary cortical region, but not from the vicinity of the injected sperm head. The Ca2+ wave became more pronounced and propagated across the egg faster in the later Ca2+ responses. An artifactual [Ca2+]i rise was inevitably produced during the ICSI procedure. The larger artifact affected the subsequent first Ca2+ response, resulting in the faster [Ca2+]i rise (time to peak, 10-20 s), slight spatial heterogeneity of [Ca2+]i rise in the ooplasm (but not a wave) and the shorter duration (3-4 min). The artifact slightly affected the amplitude of the second Ca2+ response, but little affected the later Ca2+ responses. It is suggested that the factor(s) that leaked out of the injected spermatozoon diffuses to a wide area and sensitizes Ca2+ channels of the endoplasmic reticulum to induce Ca2+ release synchronously over the ooplasm. The enhanced sensitization leads to propagating Ca2+ release initiated from the cortex that is more sensitive to the sperm factor.