ACTIVATION OF PORCINE OOCYTES VIA AN EXOGENOUSLY INTRODUCED RAT MUSCARINIC M1 RECEPTOR

Thirty hours after the beginning of in vitro maturation, porcine oocyt es were microinjected with mRNA coding for the rat muscarinic M1 recep tor. They were then incubated for 15 h to allow sufficient time for co mpleting maturation, translation of the mRNA, and insertion of the rec eptor into the plasmamembrane. They were then treated with acetylcholi ne, the receptor's agonist, and its effect on inducing various activat ion-related changes was examined. Acetylcholine treatment triggered th e release of Ca2+ from internal stores that could be blocked by atropi ne, the receptor's antagonist. The Ca2+ release was probably mediated via a G protein, since prior injection of guanosine 5'-O-(2-thiodiphos phate) (GDP-beta-S) totally inhibited the effect of the agonist. Pertu ssis toxin (PT) had no effect on the Ca2+ transients induced by acetyl choline, suggesting that the signal transduction pathway involved a PT -insensitive G protein. Electron microscopy revealed that in the injec ted oocytes, acetylcholine induced cortical granule exocytosis. The oo cytes were released from meiotic arrest as evidenced by the decrease i n H1 kinase activity measured in the oocytes during the histone H1 kin ase assay. After resuming meiosis they entered interphase: 58.8% of th e injected oocytes formed pronuclei after incubation with the agonist. Injection without subsequent acetylcholine treatment, or acetylcholin e incubation without prior injection with the receptor mRNA, did not c ause these changes. The results provide further evidence that the comp onents of a G protein-mediated signal transduction pathway exist in po rcine oocytes and that the activation of this pathway via an exogenous ly supplied G protein-coupled receptor results in a full complement of oocyte activation events. Whether this pathway transduces the activat ing signal at sperm-induced oocyte activation requires further examina tion.