Changes in organization of the endoplasmic reticulum during Xenopus oocyte maturation and activation.

The organization of the endoplasmic reticulum (ER) in the cortex of Xenopus oocytes was investigated during maturation and activation using a green fluorescent protein chimera, immunofluorescence, and electron microscopy. Dense clusters of ER developed on the vegetal side (the side opposite the meiotic spindle) during maturation. Small clusters appeared transiently at the time of nuclear envelope breakdown, disappeared at the time of first polar body formation, and then reappeared as larger clusters in mature eggs. The appearance of the large ER clusters was correlated with an increase in releasability of Ca(2+) by IP(3). The clusters dispersed during the Ca(2+) wave at activation. Possible relationships of ER structure and Ca(2+) regulation are discussed.

Sperm extract injection into ascidian eggs signals Ca(2+) release by the same pathway as fertilization.

Injection of eggs of various species with an extract of sperm cytoplasm stimulates intracellular Ca(2+) release that is spatially and temporally like that occurring at fertilization, suggesting that Ca(2+) release at fertilization may be initiated by a soluble factor from the sperm. Here we investigate whether the signalling pathway that leads to Ca(2+) release in response to sperm extract injection requires the same signal transduction molecules as are required at fertilization. Eggs of the ascidian Ciona intestinalis were injected with the Src-homology 2 domains of phospholipase C gamma or of the Src family kinase Fyn (which act as specific dominant negative inhibitors of the activation of these enzymes), and the effects on Ca(2+) release at fertilization or in response to injection of a sperm extract were compared. Our findings indicate that both fertilization and sperm extract injection initiate Ca(2+) release by a pathway requiring phospholipase C gamma and a Src family kinase. These results support the hypothesis that, in ascidians, a soluble factor from the sperm cytoplasm initiates Ca(2+) release at fertilization, and indicate that the activating factor from the sperm may be a regulator, directly or indirectly, of a Src family kinase in the egg.

Calcium release at fertilization of Xenopus eggs requires type I IP(3) receptors, but not SH2 domain-mediated activation of PLCgamma or G(q)-mediated activation of PLCbeta.

Elevation of intracellular Ca2+ at fertilization is essential for the initiation of development in the Xenopus egg, but the pathway between sperm-egg interaction and Ca2+ release from the egg's endoplasmic reticulum is not well understood. Here we show that injection of an inhibitory antibody against the type I IP(3) receptor reduces Ca2+ release at fertilization, indicating that the Ca2+ release requires IP(3). We then examine how IP(3) production is initiated. Xenopus eggs were injected with specific inhibitors of the activation of two phospholipase C isoforms, PLCgamma and PLCbeta. The Src-homology 2 (SH2) domains of PLCgamma were used to inhibit SH2-mediated activation of PLCgamma, and an antibody against G(q) family G-proteins was used to inhibit G(q)-mediated activation of PLCbeta. Though the PLCgamma SH2 domains inhibited platelet-derived growth factor (PDGF)-induced Ca2+ release in eggs with exogenously expressed PDGF receptors, they did not inhibit the Ca2+ rise at fertilization. Similarly, the G(q) family antibody blocked serotonin-induced Ca2+ release in eggs with exogenously expressed serotonin 2C receptors, but not the Ca2+ rise at fertilization. A mixture of PLCgamma SH2 domains and the G(q) antibody also did not inhibit the Ca2+ rise at fertilization. These results indicate that Ca2+ release at fertilization of Xenopus eggs requires type I IP(3)-gated Ca2+ channels, but not SH2 domain-mediated activation of PLCgamma or G(q)-mediated activation of PLCbeta.

Coordination of cellular events that precede reproductive onset in Acetabularia acetabulum: evidence for a 'loop' in development.

Amputated apices from vegetative wildtype cells of the uninucleate green alga Acetabularia acetabulum can differentiate a reproductive structure of 'cap' in the absence of the nucleus (Hämmerling, J. (1932) Biologisches Zentralblatt 52, 42-61). To define the limits of the ability of wildtype cells to control reproductive differentiation, we determined when during development apices from wildtype cells first acquired the ability to make a cap in the absence of the nucleus and, conversely, when cells with a nucleus lost the ability to recover from the loss of their apices. To see when the apex acquired the ability to make a cap without the nucleus, we removed apices from cells varying either the developmental age of the cells or the cellular volume left with the apex. Cells must have attained the adult phase of development before the enucleate apex could survive amputation and make a cap. Apices removed from cells early in adult growth required more cell volume to make a cap without the nucleus than did apices removed from cells late in adult growth. To define the limits of the cell to recapitulate development when reproduction falters, we analyzed development in cells whose caps either had been amputated or had spontaneously aborted. After loss of the first cap, cells repeated part of vegetative growth and then made a second cap. The ability to make a second cap after amputation of the first one was lost 15-20 days after cap initiation. Our data suggest that internal cues, cell age and size, are used to regulate reproductive onset in Acetabularia acetabulum and add to our understanding of how reproduction is coordinated in this giant cell.