PARTICIPATION OF INOSITOL TRISPHOSPHATE AND RYANODINE RECEPTORS IN BUFO ARENARUM OOCYTE ACTIVATION

AJMAT, M.T., BONILLA F.; ZELARAYÁN, L.; OTERINO, J.; BÜHLER; M.I.

ZYGOTE

CAMBRIDGE UNIV PRESS

Lugar: CAMBRIGDE; Año: 2010 vol. 19 p. 171 - 180

0967-1994

During fertilization, sperm entry triggers an increase in egg cytosolic Ca2+, which in mammals is followed by sustained oscillations in intracellular Ca2+ concentration ([Ca2+ ]i ) (Whitaker and Swann, 1993; Swann and Ozil, 1994). In lower species, such as echinoderms and frogs, a single transient occurs that traverses the egg in a wave-like fashion. (Nuccitelli, 1991). These transient increases in [Ca2+ ]i are essential for cortical granule release, meiosis resumption and pronuclear formation (Schultz and Kopf, 1995). A similar effect is brought about by treatment with several agents, which elevate intracellular calcium levels. Mimicking the fertilization calcium transients and oscillations has been widely used to achieve the artificial activation of oocytes in nuclear tranplantation (Collas et al, 1993; Rickords and White, 1993). The source of Ca2+ for the initial transient originates mainly from intracellular stores located in specialized regions of the endoplasmic reticulum (Miyazaki et al., 1993; Deguchi et al.,1996). Despite the importance of Ca2+ in oocyte activation, little is known about the mechanisms by which sperm causes Ca2+ release from intracellular stores. The most accepted theories involve two families of intracellular calcium channels: the inositol trisphosphate receptor (IP3R), which is universal and mediates IP3-induced- Ca2+-release (IICR) and the species-specific ryanodine receptor (RyR), which takes part in Ca2+-induced- Ca2+-release (CICR). The way in which these two pathways interact in eggs expressing both receptors is unsolved. Functional IP3Rs are present in the eggs of all studied species (Clapper and Lee, 1985; Carroll and Swann, 1992; Fissore et al., 1992; Fissore and Robl, 1993; Galione et al., 1993; Rickords and White, 1993; Swann, 1992; White et al., 1993) and appear to play a crucial role in egg activation. These receptors have been characterized in both sea urchin (Lee, 1993) and Xenopus eggs (Parys et al, 1992; 1994). Functional RyR/channel complexes appear to be present in the eggs of some species such as sea urchins, mice, bovines and humans (Galione et al.,1993; Swann,1992; Ayabe et al., 1995; Yue et al., 1995; Herbert et al.,1997), even if their role in Ca2+ signalling at fertilization is unclear (Galione et al., 1993; Ayabe et al., 1995; Berridge, 1996). Surprisingly, RyRs have not been previously detected in Xenopus laevis oocytes (Erickson et al, 2004), although their presence in oocytes from both lower and higher order species has been reported. The effect of thimerosal on IP3R-mediated Ca2+ release is well documented. Studies on several cell types demonstrate that thimerosal sensitizes IP3Rs by increasing affinity for IP3 binding (Bootman et al., 1992; Bird et al., 1993; Hilly et al., 1993). However, this action is specific only when low doses of thimerosal are used. At higher doses and during prolonged exposure, it inhibits Ca2+ -ATPase pumps. This causes a passive movement of free Ca2+ into the cytoplasm which can in turn trigger oscillations through a CICR mechanism (Herbert et al., 1997). Heparin is a well known IP3R antagonist, widely used in search of IP3Rs in many species. RyR agonists include exogenous ryanodine and caffeine, and endogenous cyclic ADP-ribose (c-ADP), a CICR modulator that has been proposed as second messenger (Berridge, 1993; Galione et al., 1993). RyR antagonists include Ruthenium Red and procaine. The aim of this work was to investigate the existence of these receptors in in vitro matured Bufo arenarum oocytes and their probable role in triggering egg activation events.