Differential contribution of cytoplasmic Ca2+ and Ca2+ influx to gamete fusion and egg activation in maize

Abstract

In multicellular organisms, gamete fusion triggers a set of events, collectively known as egg activation, that leads to the development of a new individual. Every species that has been studied shows at least one rise in cytoplasmic Ca2+ concentration ([Ca2+]Cyt) after gamete fusion which is believed to be involved in activation. Yet the source and regulation of this Ca2+ signal and the way it is transduced inside the zygote are controversial. In higher plants, in vitro fertilization (IVF) has enabled the description of a rise in [Ca2+]Cyt (ref. 4) that is sufficient for activation, and of a Ca2+ influx that spreads as a wavefront from the fusion site The relationship between these two responses is unknown. Using a new combination of methods that simultaneously monitor the extracellular flux with a Ca2+-vibrating probe, and [Ca2+]Cyt by widefield imaging, we directly determined that the Ca2+ influx precedes the [Ca2+]Cyt elevation by 40-120 s. In addition, results from experiments using the Ca2+-channel inhibitor gadolinium (Gd3+) suggest that the Ca2+ influx may be necessary for sperm incorporation. We also present evidence for a putative sperm-dependent Gd3+-insensitive localized Ca2+ influx confined to the fusion point.

Experimental Analysis of the Fertilization Process

“…Despite the blocked Ca 21 influx, however, the cytoplasmic calcium increase still occurs, presumably because of calcium release from intracellular stores. Together with the cytoplasmic calcium increase, egg contraction and cell wall deposition are observed in Gd 31 -treated cells (Antoine et al, 2001b), indicating that calcium influx is not needed to create an increased [Ca 21 ] cyt . The observed calcium influx instead may be needed for sperm incorporation and subsequent karyogamy.…”

“…This increase in [Ca 21 ] cyt is believed to be essential to egg activation and occurs at the very onset of embryo development (Stricker, 1999). In maize, combining the use of a calcium-specific vibrating probe and the single-wavelength calcium fluorochrome Fluo-3, Antoine et al (2001b) demonstrated that gamete fusion always triggers a calcium influx, which is then followed by an increase in cytoplasmic calcium ( Figures 4A and 4B). The site of the calcium influx is initiated at the fusion site and within a few minutes moves in a wave-like pattern over the rest of the membrane, resulting in a homogeneous influx (Antoine et al, 2000).…”

“…The observed calcium influx instead may be needed for sperm incorporation and subsequent karyogamy. These observations allow flowering plants to be included in the general model of multicellular Adapted from Antoine et al (2001b) and reproduced with the permission of the Nature Publishing Group.…”

A novel in vitro system for gamete fusion in maize

“…The results indicated that adding calcium was not necessary for gamete fusion with the presence of BSA in the medium, although the low concentration of calcium at micromorlar level might benefit gametes for their adhesion and fusion. It was reported that maize gamete fusion could trigger a calcium influx, which was then followed by an increase of cytoplasmic calcium [21]. In this process, the extracellular calcium that penetrated the egg cells may partly contribute to the cytosolic calcium increase.…”

Formation of triploid plants via possible polyspermy

“…[23][24][25][26] It has been indicated that polyspermy block functions in the egg cell [27][28][29][30] and the central cell 30 to promote faithful double fertilization 31 . In addition, Ca 2C -dependent egg activation occurs in plants as well as animals, [32][33][34] and it has been postulated that the increased Ca 2C level triggers the polyspermy block of fertilized egg cells via Ca…”