LOCALIZATION AND REGULATION OF RYANODINE RECEPTOR IN BOVINE OOCYTES

We have previously reported that injection of ryanodine receptor agoni sts into mature bovine oocytes induces intracellular calcium release, Indicative of the existence of ryanodine receptors. In this experiment , further evidence of the ryanodine receptor localization, and develop mental regulation in bovine oocytes is presented. The possible physiol ogical significance is also suggested, Using a rabbit antibody against the rabbit cardiac muscle ryanodine receptor, the ryanodine receptor was observed uniformly localized in the periphery of mature bovine ooc ytes, while a weak and discontinuous signal was observed in the germin al vesicle intact stage of bovine oocytes, As oocytes progress to the metaphase I stage, the ryanodine receptor localization became more int ense and continuous, yet not comparable to that observed in the metaph ase II oocytes. These modifications correlate with the intracellular c alcium responsiveness to ryanodine, A 200-mu M injection of ryanodine induces a low intracellular calcium transient in germinal vesicle-stag e bovine oocytes, while peaked intracellular calcium transient are sub sequently observed in the metaphase II-stage oocytes. However, no sign ificant changes in the amplitude of intracellular calcium transients i nduced by 250 nM inositol 1,4,5-trisphosphate and 10 mu M ionomycin we re observed in oocytes at a comparable stage, Fertilization induced a significant decrease in ryanodine receptor signal; similar changes wer e also observed in oocytes injected with 200 mu M ryanodine or incubat ed with 10 mu M ionomycin. However, no changes in ryanodine signal wer e observed in oocytes injected with vehicle medium. Furthermore, injec tion of either ryanodine or inositol 1,4,5-trisphosphate induced subse quent pronuclear formation and cleavage. These data indicate that the ryanodine receptor is closely regulated and associated with early cell ular changes following fertilization; stimulation of this receptor res ults in the activation of bovine oocytes, and it is likely that this r eceptor may play a role at fertilization.