Wave of free calcium at fertilization in the sea urchin egg visualized with fura‐2

Hafner, Mathias; Petzelt, Christian; Nobiling, R.; Pawley, James B.; Kramp, Douglas C.; Schatten, Gerald

doi:10.1002/cm.970090309

Cited by 70 publications

(32 citation statements)

References 29 publications

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“…This value of the increased Ca^coincided with those of previous reports (9, 25, 32, 33). When a CaEGTAbuffer solution, which increases the Caj, and an IP3 solution, which induces Ca2+ release and then increases the Cai, were microinjected into the eggs, the change in Caj was also measured and compared with that upon fertilization.…”

supporting

confidence: 93%

Propagation of Transient Ca2+ Increase in Sea Urchin Eggs upon Fertilization and Its Regulation by Microinjecting EGTA Solution.

Mohri

Hamaguchi

1991

Cell Struct. Funct.

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ABSTRACT. Upon fertilization, the concentration of intracellular Ca2+ (CaO in sea urchin eggs increased up to 3 juM when measured with fura-2, a fluorescent Ca indicator and the increase in Cai traversed from the sperm entry point as a waveover the entire egg at the meanpropagation velocities of 5.0 /um/sec in C. japonicus egg and 5.3 //m/sec in H. pulcherrimus egg. However, the velocity was not uniform; i.e., it was rapid in the vicinity of the sperm entry point and the opposite point, but slow in the central region of the egg. Microinjecting a CaEGTAbuffer and an IP3 solution into the C. japonicus egg induced the transient Cai increase more rapidly than that upon fertilization, due perhaps to the diffusion of the injectates. In order to investigate Ca2+ release during Cai increase upon fertilization, EGTAsolutions were microinjected into un fertilized or fertilizing eggs. Microinjecting 100 mMEGTA(final concentration of 1 mM)not only suppressed the transient Cat increase, but also reduced the increased Cai rapidly, and never induced egg activation after insemination, whereas 10 mMEGTA(final concentration of 0.1 mM)did not significantly affect the Cai increase or the activation. Ca2+ released upon fertilization was estimated to be 150-170 juM in the egg cytoplasm from the amount of microinjected EGTAand fura-2. It was concluded that although more than 150 juM of Ca2+ was released intracellularly upon fertilization, Cai increased to only a few juM because most of the released Ca2+ was sequestered by intracellular Ca2+ binding substances.Upon fertilization, a transient increase in intracellular Ca2+concentration in the egg cytoplasm (CaO is one of the most universal events (7, 19, 26, 32). The transient Caj increase has been reported in the oocytes or eggs ofmedaka (7,17, 30, 38), Xenopus (1, 21), golden hamster (22), mouse (3, 4), ascidian (31), starfish (2, 6), and seaurchin (5,9,10, 25, 32, 33, 37, 38). In seaurchin eggs, the peak values of the increase in Ca{ were previously reported to be 2.5-9juM (9, 25, 32, 33). The transient Cai increase is a key point for the development of the egg. When the increase is blocked by microinjection of EGTA solutions, fertilization never succeeds even when sperm enter the egg (ll, 12). In contrast, the Cai raised artificially by microinjecting Ca2+ or second messengers into the un fertilized egg results in egg activation (ll, 23, 35). In large oocytes such as Medaka and Xenopusoocytes, the transient C^increase propagates from the sperm entry point to the antipode of the egg in a ring shape (7,17, 30, 38). However, in small eggs such as echinoderm eggs, although the increase starts near the sperm entry point and propagates over the en- tire egg, the precise process of the Caj change has not yet been investigated in terms of where and how the change in Cai occurs (9, 10). In addition, the propagation velocity of the Cai increase has not yet been analyzed. Moreover, the amount of Ca2+ released intracellularly upon fertilization has not been investigated. In the present study, t...

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supporting

confidence: 93%

Propagation of Transient Ca2+ Increase in Sea Urchin Eggs upon Fertilization and Its Regulation by Microinjecting EGTA Solution.

Mohri

Hamaguchi

1991

Cell Struct. Funct.

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“…A transient Ca2+ increase was also reported to propagate through the egg cytoplasm (5,6,22). Therefore, CGBmaypropagate at the same velocity of a transient Ca2+ increase upon fertilization.…”

Section: Discussionmentioning

confidence: 97%

Quantitative analysis of the process and propagation of cortical granule breakdown in sea urchin eggs.

Mohri

Hamaguchi

1990

Cell Struct. Funct.

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ABSTRACT. Cortical granule breakdown in sea urchin eggs has been investigated with a video microscope system using Nomarski differential interference contrast optics, when induced by fertilization, microinjecting inositol 1, 4, 5-trisphosphate (IP3) or Ca-EGTAbuffer solution into the egg, or per fusing a mediumcontaining 1 mM Ca2+ to isolated cortices. The cortical granule increased up to 1.2 times in diameter and broke down within 40 msec. These values were almost constant amongthe three methods used to induce cortical granule breakdown. Upon fertilization, the cortical granule breakdown propagated over the egg surface at a speed of 3.3 jum/sec in Clypeaster japonicus eggs, which indicates that cortical granule breakdown propagated through the 3.3-jumwide egg surface within 1 sec. In such a small area of the egg surface, however, it took muchmore than 1 sec for all cortical granules to break down because the maximal rate of breakdown was 7.6%Vsec; that is, it took 9 sec and 18 sec for 50% and 90%, respectively, of cortical granules to break down. Moreover, the rate did not simply decrease with time, and a shoulder was found during the reducing phase, which suggests that cortical granules are divided into fast and slow breakdown groups according to the responsiveness to the breakdown stimulus. The cortical granule breakdown induced by microinjecting the Ca-EGTAbuffer and IP3 solutions propagated at 68 //m/sec and 35 //m/sec, respectively. The stimulus for cortical granule breakdown is discussed concerning the transient intracellular Ca2+ increase.

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“…These measurements and the calibration of Fura-2 fluorescence was performed according to standard protocols (31). Briefly, cells were loaded for 1 h with 5 M Fura-2 AM diluted in Fluoronic R F-127.…”

Section: Calpainolysis Of Bcl-2 Proteins In Vitromentioning

confidence: 99%

Ionomycin-activated Calpain Triggers Apoptosis

Gil-Parrado¹,

Fernández-Montalván²,

Assfalg-Machleidt³

et al. 2002

Journal of Biological Chemistry

182

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Ubiquitous calpains (-and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitro both ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-x L at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. We conclude that ionomycin-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway.

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Wave of free calcium at fertilization in the sea urchin egg visualized with fura‐2

Cited by 70 publications

References 29 publications

Propagation of Transient Ca2+ Increase in Sea Urchin Eggs upon Fertilization and Its Regulation by Microinjecting EGTA Solution.

Propagation of Transient Ca2+ Increase in Sea Urchin Eggs upon Fertilization and Its Regulation by Microinjecting EGTA Solution.

Quantitative analysis of the process and propagation of cortical granule breakdown in sea urchin eggs.

Ionomycin-activated Calpain Triggers Apoptosis

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