MAINTENANCE OF BOVINE OOCYTES IN PROPHASE OF MEIOSIS-I BY HIGH [CAMP](I)

The effects of high intracellular cAMP concentrations ([cAMP](i)) on g erminal vesicle maintenance of bovine cumulus-oocyte complexes were in vestigated, using 8-bromo-3',5'-cAMP (8-Br-cAMP) or an invasive adenyl ate cyclase from Bordetella pertussis to increase the [cAMP](i). The e ffects of interactions of these agents with macromolecular supplements in culture medium (fetal calf serum, FCS; polyvinylpyrrolidone, PVP; BSA), and different methods of processing complexes before culture, on subsequent germinal vesicle maintenance by invasive adenylate cyclase were studied. While 8-Br-cAMP was unable to maintain germinal vesicle arrest in the majority of oocytes for 20 h (36% with FCS, 24% with BS A, 18% with PVP), it maintained germinal vesicle arrest in a high prop ortion of cumulus-enclosed oocytes when BSA or PVP was used (37% with FCS, 52% with BSA, 53% with PVP). The difference in frequency of germi nal vesicle maintenance between macromolecular supplements was not rel ated to [cAMP](i) when assayed after culture for 2 h with invasive ade nylate cyclase. Complexes processed in whole follicular fluid were not maintained in meiotic arrest (26%) when cultured with invasive adenyl ate cyclase and PVP. Complexes processed in follicular fluid with 3-is obutyl I-methylxanthine (IBMX) plus invasive adenylate cyclase were ar rested at the germinal vesicle stage at high frequencies (65%), while those processed in IBMX or IBMX plus 8-Br-cAMP-supplemented follicular fluid had intermediate (43% and 49%, respectively) frequencies of int act germinal vesicles. Oocyte complexes processed in follicular fluid supplemented with IBMX and invasive adenylate cyclase formed morulae a nd blastocysts (27.2%), as did oocytes processed in follicular fluid a lone (26%). Phosphoprotein profiles showed that control oocytes and 8- Br-cAMP-treated oocytes share a profile that is different from that of oocytes treated with invasive adenylate cyclase. These results show t hat increased [cAMP], reversibly maintains bovine oocytes in meiotic a rrest for an extended period without the occurrence of the post-transl ational protein modifications observed during meiotic resumption or tr ansient arrest.