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

Abstract

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...

Cortical granule exocytosis is triggered by different thresholds of calcium during fertilisation in sea urchin eggs

“…Others have reported that the cortical granules are not sensitive to calcium concentrations below 10 uM and that the cortical granule population has a submaximal response to any calcium concentration under 100 uM (Vogel et al, 1996). Although many have estimated peak free calcium concentrations to be around 1 uM (Eisen et al, 1984;Hafner et al, 1988), some estimates have the released calcium increasing to as much as 50-170 uM (Zucker & Steinhardt, 1978;Mohri & Hamaguchi, 1991). Although many have estimated peak free calcium concentrations to be around 1 uM (Eisen et al, 1984;Hafner et al, 1988), some estimates have the released calcium increasing to as much as 50-170 uM (Zucker & Steinhardt, 1978;Mohri & Hamaguchi, 1991).…”

Cyclic ADP-ribose-gated Ca2+ Release in Sea Urchin Eggs Requires an Elevated [Ca2+]

“…Calmodulin, a required cofactor for cADPr-induced Ca 2ϩ release in sea urchin eggs (18 -20), is a possible candidate for mediating Ca ], and about 60% of maximum activity at 100 nM [Ca 2ϩ ]) in egg extracts appears inconsistent with our estimate that cADPr activity in the intact egg was less than 1% of maximum at normal resting [Ca 2ϩ ] (measured by others to be in the 150 -200 nM range (22,23) Recently, Genazzani et al (35) examined the kinetics of cADPr-induced Ca 2ϩ release in sea urchin egg homogenates as a function of the cADPr concentration using a stop flow apparatus with a 25-ms time resolution. They reported an apparent acceleration of release in response to low, submaximal levels of cADPr, which they attributed to a Ca 2ϩ -dependent facilitation.…”

Calcium signalling in early embryos

“…In echinoderms, the microinjection of calcium can trigger a propagating calcium wave (Hamaguchi & Hiramoto 1981;Mohri & Hamaguchi 1991) and ruthenium red, an RyR antagonist, reduces the propagation velocity of the wave (Miyazaki 1988;Galione et al 1993). A calcium wave can be triggered in sea urchin eggs by calcium influx after sensitizing the RyR .…”