THE EXISTENCE OF INOSITOL 1,4,5-TRISPHOSPHATE AND RYANODINE RECEPTORSIN MATURE BOVINE OOCYTES

Intracellular Ca2+ (Ca-i(2+)) transients during fertilization are crit ical to the activation of eggs in all species studied, Activation of b oth the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and ryanodi ne receptor (RYR) are responsible for the calcium oscillations during fertilization in sea urchin eggs, Using in vitro matured bovine oocyte s loaded with Fura-2 AM ester as Ca-i(2+) indicator, we addressed whet her IP3Rs and RYRs coexist in mammalian eggs, Our results indicate tha t microinjection of 50-250 nM IP3 or 10-20 mM caffeine, 100-200 mu M r yanodine and 4-8 mu M cyclic ADP-ribose all induced Ca-i(2+) release. The Ca-i(2+) release induced by 250 nM IP3 could only be inhibited by prior injection of 1 mg/ml heparin which was overcome by continuous in jection of IP3 to 1 mu M. Prior injection of either 50 mu M ruthenium red, 50 mu M procaine or 1% vehicle medium (VM) did not affect the Ca- i(2+) release induced by IP3. Prior injection of heparin or VM did not affect the Ca-i(2+) release induced by 10-20 mM caffeine or 200 mu M ryanodine, but prior injection of 50 mu M ruthenium red or procaine co mpletely inhibited the effect of 10-20 mM caffeine, In addition, conti nuous injection of caffeine up to 40 mM overcame the inhibitory effect of ruthenium red or procaine, The same 50 mu M concentration of ruthe nium red or procaine only partially blocked the effect of 200 mu M rya nodine, but 200 mu M ruthenium red or procaine completely blocked the effect of 200 mu M ryanodine, Oocytes were refractory for 15 minutes t o further injections of IP3 after the initiation of Ca-i(2+) release i nduced by 200 nM IP3; a 10 minute refractory period was observed for 1 0-20 mM caffeine and 200 mu M ryanodine; either caffeine or ryanodine can desensitize RYRs to the other. However, the desensitization of IP3 Rs by 200 nM IP3 does not abolish the effect of 200 mu M ryanodine, as effectively as the reciprocal treatment, Prior injection of a subthre shold concentration of ryanodine itself only induced a slight increase in Ca-i(2+) level, but it sensitized the RYR to a subsequent injectio n of a subthreshold concentration of caffeine, Similar results were ob tained when ryanodine was first injected followed by injection of caff eine Based on these results, we conclude that independent IP3Rs and RY Rs exist in mature bovine oocytes.