| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1997: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1996: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
Sperm-induced activation of mammalian eggs is associated with a transient increase in Ca^<2+> 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 Ca^<2+> 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 Ca^<2+> store, I 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 M_r=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-i
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ntact 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 post-fertilization form ZP2_f 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. Last, treatment of eggs under conditions that would block the release of Ca^<2+> 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 Ca^<2+> from this store is not essential for sperminduced mouse egg activation. Less
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