Demonstration of calcium uptake and release by sea urchin egg cortical endoplasmic reticulum.

Abstract

Abstract. The calcium indicator dye fluo-3/AM was loaded into the ER of isolated cortices of unfertilized eggs of the sea urchin Arbacia punctulata . Development of the fluorescent signal took from 8 to 40 min and usually required 1 mM ATP The signal decreased to a minimum level within 30 s after perfusion with 1 FAM InsP3 and increased within 5 min when InsP3 was replaced with 1 mM ATP. Also, the fluorescence signal was lowered rapidly by perfusion with 10 p,M NTRACELLULAR free calcium rises near the sperm entry point in the eggs of many animals ; a wave of elevated calcium then proceeds from this region by a release of calcium from internal stores (Gilkey et al ., 1978;Jaffe, 1983;Hafner et al., 1988). The calcium wave is crucial in the activation of the egg (Whitaker and Steinhardt, 1982;Jaffe, 1983) ; for instance, its passage triggers the postfertilization wave ofcortical granule fusion (Hamaguchi and Hiramoto, 1981) .In the sea urchin egg cortex preparation, the former interior of the egg surface is exposed and amenable to experimentation (Vacquier, 1975). This preparation retains some features of normal calcium regulation. Isolated cortices sequester 'SCa in the presence of ATP, and release preloaded°SCa in response to inositol 1,4,5 trisphosphate (InsP3) ' (Oberdorf et al ., 1986;Payan et al ., 1986) . However, an important issue that has not been resolved by the above isotope studies is the identity of the membrane compartments that sequester and release calcium . Several organelles are present on the sea urchin cortex, including cortical granules (Vacquier, 1975), pigmented and acidic vesicles (Sardet, 1984), and the ER (Sardet, 1984;Chandler, 1984) .Recently, a calsequestrin-like protein has been isolated from sea urchin eggs (Oberdorf et al., 1988), and an antibody to this protein labels the cortical ER (Henson et al., 1989) . In striated muscle, calsequestrin is a low affinity, high capacity calcium binding protein; the presence of a similar calcium binding protein in the ER of sea urchin eggs suggests that the ER is capable of sequestering calcium, but direct evidence for calcium uptake and release from the ER is lacking. A23187 or 10 pM ionomycin . These findings demonstrate that the cortical ER is a site of ATP-dependent calcium sequestration and InSP3-induced calcium release. A light-induced wave of calcium release, traveling between 0.7 and 2.8 pm/s (average speed 1.4 j am/s, N = 8), was sometimes observed during time lapse recordings ; it may therefore be possible to use the isolated cortex preparation to investigate the postfertilization calcium wave.We report here that the fluorescent calcium indicator dye, fluo-3 (Minta et al ., 1989), when applied in its AM ester form, produces a signal from the cortical ER ofcortices prepared from Arbacia punctulata eggs. Once present in the ER, the dye can be used to determine whether the ER sequesters or releases calcium . Materials and Methods Sea Urchin EggsEggs were obtained from Arbacia punctulata by injection of 0.5 M KCl into the coelomic cavity. ...

Calcium permeability of the neuronal nuclear envelope: evaluation using confocal volumes and intracellular perfusion

“…7) suggests that part of the cytoplasmic fluo 3 signal is not responding to changes in the cytoplasmic concentration of divalent cations. Fluo 3 inside organelles may be saturated with calcium (Chandra et al, 199 1;Krause, 199 1;Terasaki and Sardet, 1991;Glennon et al, 1992) and would therefore constitute a substantial fluorescence signal that is insensitive to changes in cytosolic calcium.…”

Endoplasmic reticulum reorganizations and Ca2+ signaling in maturing and fertilized oocytes of marine protostome worms: the roles of MAPKs and MPF

“…In addition to changes that occur in the nucleus during GVBD, oocyte maturation can also trigger marked alterations in cytoplasmic organelles. For example, the endoplasmic reticulum (ER), which most probably represents the major internal store of Ca 2+ to be mobilized at fertilization (Eisen and Reynolds, 1985;Han and Nuccitelli, 1990;Terasaki and Sardet, 1991), develops discrete aggregates (i.e. 'clusters' or 'microdomains') in the maturing oocytes of several animal groups that have been investigated (Kline, 2000;Sardet et al, 2002).…”

Molecular Characterization of the Starfish Inositol 1,4,5-Trisphosphate Receptor and Its Role during Oocyte Maturation and Fertilization

“…Nicotinic acid adenine dinucleotide phosphate is also known to release Ca 2ϩ from internal stores in sea urchin eggs (4,5) and starfish eggs (6), but the molecular mechanism is still unknown. Several observations suggest that IP 3 mediates Ca 2ϩ release during fertilization: (i) phosphatidylinositol lipid turnover and IP 3 levels increase rapidly during fertilization (7)(8)(9)(10)(11), (ii) IP 3 injection of unfertilized eggs and IP 3 application to endoplasmic reticulum isolated from eggs causes calcium release (12)(13)(14)(15)(16)(17)(18), (iii) injection of phospholipase C-␥ Src homology 2 domain into oocyte inhibits Ca 2ϩ release during fertilization by inhibiting the generation of IP 3 (19), and (iv) injection of oocytes with heparin, a nonspecific competitive inhibitor of IP 3 R, causes abnormal fertilizationinduced calcium dynamics in a dose-dependent fashion (20 -24). However, the effects of heparin are difficult to interpret because of its lack of specificity.…”