Regulation of mouse egg activation: presence of ryanodine receptors and effects of microinjected ryanodine and cyclic ADP ribose on uninseminated and inseminated eggs

Abstract

Sperm-induced activation of mammalian eggs is associated with a transient increase in Ca2+ concentrations thought to be derived from inositol 1,4,5-trisphosphate-sensitive and -insensitive intracellular stores. Whereas the importance of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores has been evaluated, the identity and role of inositol 1,4,5-trisphosphate-insensitive stores are poorly understood. To explore the role of the ryanodine-sensitive Ca2+ store, we first used reverse transcription-polymerase chain reaction to identify transcripts of the ryanodine receptor in eggs and determined that transcripts for the type 2 and 3 receptor were present. Immunoprecipitation of radioiodinated egg extracts with an antibody that recognizes both type 2 and 3 receptors detected specifically a band of Mr = 520,000. Immunolocalization of the receptor(s) using laser-scanning confocal microscopy revealed that the receptor(s) was uniformly distributed in the cortex of the germinal vesicle-intact oocyte, but became asymmetrically localized to the cortex in a region apposed to the meiotic spindle in the metaphase II-arrested egg; this asymmetrical localization developed by metaphase I. The role of the ryanodine receptor in mouse egg activation was examined by determining the effects of microinjected ryanodine or cyclic ADP ribose on endpoints of egg activation in either uninseminated or inseminated eggs. Ryanodine induced the conversion of the zona pellucida glycoprotein ZP2 to its postfertilization form ZP2f in a biphasic concentration-dependent manner; nanomolar concentrations stimulated this conversion, whereas micromolar concentrations had no stimulatory effect. Cyclic ADP ribose also promoted the ZP2 conversion, but with a hyperbolic concentration dependence. Neither of these compounds induced cell cycle resumption. Inhibiting the inositol 1,4,5-trisphosphate-sensitive Ca2+ store did not inhibit the ryanodine-induced ZP2 conversion and, reciprocally, inhibiting the ryanodine-sensitive Ca2+ store did not inhibit the inositol 1,4,5-trisphosphate-induced ZP2 conversion. Last, treatment of eggs under conditions that would block the release of Ca2+ from the ryanodine-sensitive store had no effect on any event of egg activation following fertilization. Results of these experiments suggest that although ryanodine receptors are present and functional, release of Ca2+ from this store is not essential for sperm-induced egg activation.

The cortical reaction and development of activation competence in mammalian oocytes

“…To the best of our current knowledge, at fertilization, Ca release appears to be mediated principally through IP 3 in mammalian eggs (Miyazaki et al, 1993). Although functional ryanodine receptors (Ayabe et al, 1995) and ryanodine-sensitive Ca stores (Swann, 1992) are present in mouse eggs, ryanodine receptor-mediated Ca release is not required for mouse egg activation Ayabe et al, 1995).…”

“…Direct and indirect localization studies of the distribution of Ca binding and regulatory proteins are consistent with their localization in the ER. These include the IP 3 receptor, a ryanodine receptor/ Ca release channel, calsequestrin and calreticulin (Henson et al, 1989(Henson et al, , 1990McPherson et al, 1992;Kume et al, 1993;Parys et al, 1994;Ayabe et al, 1995;Shiraishi et al, 1995). IP 3 induces Ca release principally from the ER in studies of oocyte cytoplasmic fractions (Han and Nuccitelli, 1990).…”

“…The development of competence to undergo CG release (as well as egg activation) is likely to depend on not only the position of Ca stores and Ca regulatory proteins, but also their concentration and functional status. In mature mammalian eggs, intracellular Ca release is primarily controlled by a mechanism involving IP 3 -induced release Ayabe et al, 1995;Miyazaki, 1995) as described previously. In theory, this release mechanism could be developmentally gated at many points in the egg's signalling pathway, e.g.…”

Modulators of calcium signalling at fertilization

“…In ascidians, the IP3 receptor generates Ca 2+ release responsible for the fertilization-associated Ca 2+ wave, but Ca 2+ released through the ryanodine receptor promotes post-fertilization plasma membrane insertion events that do not occur when only the IP3 receptor is active [157]. Although the ryanodine receptor can be detected in mammalian eggs, it does not appear to contribute significantly to Ca 2+ release or other egg activation events at fertilization in mammals [158][159][160][161].…”

Oocyte activation after intracytoplasmic injection of mature and immature sperm cells

“…There is little doubt that IP 3sensitive stores are present in oocytes (Miyazaki et al, 1993;Kline and Kline, 1994;Fissore et al, 1995;Miyazaki, 1995). On the other hand, ryanodine-sensitive stores have been detected by some workers (Swann, 1992;Ayabe et al, 1995;Yue et al, 1995;Sousa et al, 1996a) but not by others (Miyazaki et al, 1993;Kline and Kline, 1994;Fissore et al, 1995). The presence of ryanodinesensitive calcium stores in oocytes and their role in the oocyte activation mechanism have been recently demonstrated in humans (Sousa et al, 1996a), and these calcium stores have been suggested to participate, together with IP 3 -sensitive stores, in a two-store mechanism of calcium oscillations Tesarik and Sousa, 1996).…”