The existence of inositol 1,4,5-trisphosphate and ryanodine receptors in mature bovine oocytes

Abstract

ABSTRACT Intracellular Ca2+ (Ca2+i) transients during fertilization are critical to the activation of eggs in all species studied. Activation of both the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and ryanodine receptor (RYR) are responsible for the calcium oscillations during fertilization in sea urchin eggs. Using in vitro matured bovine oocytes loaded with Fura-2 AM ester as Ca2+i indicator, we addressed whether IP3Rs and RYRs coexist in mammalian eggs. Our results indicate that microinjection of 50-250 nM IP3 or 10-20 mM caffeine, 100-200 μM ryanodine and 4-8 μM cyclic ADP-ribose all induced Ca2+i release. The Ca2+i release induced by 250 nM IP3 could only be inhibited by prior injection of 1 mg/ml heparin which was overcome by continuous injection of IP3 to 1 μM. Prior injection of either 50 μM ruthenium red, 50 μM procaine or 1 % vehicle medium (VM) did not affect the Ca2+i release induced by IP3. Prior injection of heparin or VM did not affect the Ca2+i release induced by 10-20 mM caffeine or 200 μM ryanodine, but prior injection of 50 μM ruthenium red or procaine completely inhibited the effect of 10-20 mM caffeine. In addition, continuous injection of caffeine up to 40 mM overcame the inhibitory effect of ruthenium red or procaine. The same 50 μM concentration of ruthenium red or procaine only partially blocked the effect of 200 μM ryanodine, but 200 μM ruthenium red or procaine completely blocked the effect of 200 μM ryanodine. Oocytes were refractory for 15 minutes to further injections of IP3 after the initiation of Ca2+i release induced by 200 nM IP3; a 10 minute refractory period was observed for 10-20 mM caffeine and 200 μM ryanodine; either caffeine or ryanodine can desensitize RYRs to the other. However, the desensitization of IP3Rs by 200 nM IP3 does not abolish the effect of 200 μM ryanodine, as effectively as the reciprocal treatment. Prior injection of a subthreshold concentration of ryanodine itself only induced a slight increase in Ca2+i level, but it sensitized the RYR to a subsequent injection of a subthreshold concentration of caffeine. Similar results were obtained when ryanodine was first injected followed by injection of caffeine Based on these results, we conclude that independent IP3Rs and RYRs exist in mature bovine oocytes.