Inositol 1,4,5-trisphosphate-induced calcium release in the organelle layers of the stratified, intact egg of Xenopus laevis.

Abstract

Abstract. Using double-barreled, Ca2÷-sensitive microelectrodes, we have examined the characteristics of the Ca 2+ release by inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in the various layers of Xenopus laevis eggs in which the organelles had been stratified by centrifugation. Centrifugation of living eggs stratifies the organelles yet retains them in the normal cytoplasmic milieu. The local increase in intracellular free Ca 2+ in each layer was directly measured under physiological conditions using theta-tubing, doublebarreled, Ca2+-sensitive microelectrodes in which one barrel was filled with the Ca 2+ sensor and the other was filled with Ins(1,4,5)P3 for microinjection. The two tips of these electrodes were very close to each other (3/~m apart) enabling us to measure the kinetics of both the highly localized intracellular Ca 2÷ release and its subsequent removal in response to Ins(1,4,5)P3 injection. Upon Ins(1,4,5)P3 injection, the ER-enriched layer exhibited the largest release of Ca 2+ in a dosagedependent manner, whereas the other layers, mitochondria, lipid, and yolk, released 10-fold less Ca 2÷ in a dosage-independent manner. The removal of released Ca 2÷ took place within ,x,1 min. The sensitivity to Ins(1,4,5)P3 and the time course of intracellular Ca ~+ release in the unstratified (unactivated) egg is nearly identical to that observed in the ER layer of the stratified egg. Our data suggest that the ER is the major organelle of the Ins(1,4,5)Prsensitive Ca 2÷ store in the egg of Xenopus laevis. transient increase in intracellular free calcium concentration ([Ca2+]~) during sperm-egg interaction is one of the main ionic events of fertilization that triggers the initiation of development of the zygote (13, 21,46,60). This increase in [Ca2+]i has been observed during the activation of a wide variety of eggs using at least three different techniques for Ca ~+ measurement. The photoprotein, aequorin, was the first Ca 2+ indicator to reveal a wave of increased free [Ca2+]i after activation of the eggs of the medaka fish (16,20,39,62), sea urchin (12, 48), starfish (10), frog (25), mouse (7,8), and hamster (29). Pairs of Ca ~+ electrodes were used to study the wave in frog eggs (3,4,35) and the fluorescent Ca 2+ probe, fura-2, has been used in sea urchins (17,51,59).Recent studies of sea urchin and frog eggs have indicated that sperm-egg interaction may release Ca 2+ through the inositol lipid cascade. Sperm binding to its receptor at the egg surface activates a phosphoinositide-specific phospholipase C enzyme (24,54,55). The phospholipase C enzyme hydrolyzes phosphoinositol 4,5-bisphosphate (PIP2) t producing two second messengers, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol. Ins(1,4,5)P3 releases Ca 2+ from intracellular Ca 2÷ stores whereas diacylglycerol activates the phospholipid-and Ca:÷-dependent protein kinase, protein kinase C, which is involved in many cellular re-1. Abbreviaa'ons used in this paper: CG, cortical granules; Ins(l,4,5)P3, inositol 1,4,5-trisphosphate; pCa, negative...

Calcium influx factor is synthesized by yeast and mammalian cells depleted of organellar calcium stores

“…Fig. 3A shows a photomicrograph of the centrifuged oocytes, in which [Ca 2ϩ ] i increases in response to IP 3 and thapsigargin were previously localized to the portion of the oocyte enriched for the ER (17,30). Our observations also showed maximal [Ca 2ϩ ] i increases near this band and were faithfully mimicked by a model based on a diffusible CIF produced only by the ER-enriched band (A.B., P.C., R.B.M., and J.E.K., manuscript in preparation).…”

“…After 30 min, the oocytes in some experiments were transferred to a centrifuge tube containing 5 ml 30% Ficoll 400. Stratification was carried out at 4,000 ϫ g for 1 hr (17). The oocytes were oriented on the microscope stage with the animal pole facing the objective or, for those that were centrifuged, so that all four layers of the stratified oocyte were visible in the optical plane.…”

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).…”

Cytochemical Ca2+ Distribution in Activated Discoglossus pictus Eggs: A Gradient in the Predetermined Site of Fertilization

“…In this regard, it is noteworthy that recent studies on IP3-induced Ca2+ release in stratified eggs of Xenopus laevis have demonstrated that although all organelle layers are able to release (and sequester) Ca2+, the reticulum-enriched layer releases ten fold as much Ca2+ (17).…”