Maintenance of bovine oocytes in prophase of meiosis I by high [cAMP]i.
The effects of high intracellular cAMP concentrations ([cAMP]i) on germinal vesicle maintenance of bovine cumulus-oocyte complexes were investigated, using 8-bromo-3',5'-cAMP (8-Br-cAMP) or an invasive adenylate cyclase from Bordetella pertussis to increase the [cAMP]i. The effects 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 BSA, 18% with PVP), it maintained germinal vesicle arrest in a high proportion of cumulus-enclosed oocytes when BSA or PVP was used (37% with FCS, 52% with BSA, 53% with PVP). The difference in frequency of germinal vesicle maintenance between macromolecular supplements was not related to [cAMP]i when assayed after culture for 2 h with invasive adenylate cyclase. Complexes processed in whole follicular fluid were not maintained in meiotic arrest (26%) when cultured with invasive adenylate cyclase and PVP. Complexes processed in follicular fluid with 3-isobutyl 1-methylxanthine (IBMX) plus invasive adenylate cyclase were arrested 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 intact germinal vesicles. Oocyte complexes processed in follicular fluid supplemented with IBMX and invasive adenylate cyclase formed morulae and blastocysts (27.2%), as did oocytes processed in follicular fluid alone (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 that increased [cAMP]i reversibly maintains bovine oocytes in meiotic arrest for an extended period without the occurrence of the post-translational protein modifications observed during meiotic resumption or transient arrest.
Aktas
H
H
Department of Meat and Animal Science, University of Wisconsin, Madison 53706, USA.
Wheeler
M B
MB
Rosenkrans
C F
CF
First
N L
NL
Leibfried-Rutledge
M L
ML
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000494:pharmacology; D000818:Animals; D001886:Bordetella pertussis; D002417:Cattle; D018929:Cell Culture Techniques; D003470:Culture Media; D000242:Cyclic AMP / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D009865:Oocytes / Q000166:cytology*; D011418:Prophase; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013048:Specimen Handling
D003470:Culture Media; D015124:8-Bromo Cyclic Adenosine Monophosphate; D012710:Serum Albumin, Bovine; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D015056:1-Methyl-3-isobutylxanthine