Effects of cyclic AMP on the spontaneous meiotic maturation of cumulus‐free bovine oocytes cultured in chemically defined medium

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Abstract
The rate of spontaneous meiotic maturation and the period of commitment to this process were determined in bovine oocytes devoid of surounding cumulus cells, cultured in chemically defined medium with bovine serum albumin in the absence of serum. The effects of compounds that are known to elevate levels of intracellular cyclic adenosine monophosphate (cAMP) on the resumption and progression of meiosis were investigated. Bovine oocytes were mass-harvested, dended of cumulus cells, and cultured in 2A-BMOC medium supplemented with 0.5% bovine serum albumin. Intracellular cAMP levels were indirectly modified using 8-bromo-cAMP, bibutyryl cAMP (dbcAMP), forskolin, or 3-isobutyl-1-methyl xanthine (IBMX). Meiotic maturation was scored cytogenetically. Ninety percent of denuded bovine oocytes mature after 24 h, with 65% progressing beyond anaphase I. These oocytes remain at the germinal vesicle (GV) stage for up to 8 h in culture. GV breakdown (GVBD) occurs in 40.5% of oocytes at 9 h. The peak times for the different meiotic stages were 12 h for diakinesis, 15 h for late diakinesis to metaphase I, 20 h for metaphase I, and 24 h for telophase I. By 48 h, most had reached metaphase II. There is a 2-h lag period between the time at which they become irreversibly committed to mature (at 7 h) and when they demonstrate GVBD (at 9 h). Incubation for 12 h with high concentrations for 8-bromo-cAMP and forskolin significanly inhibited GVBD, while the effect of dbcAMP was similar but less pronounced. By 24 h, most of the control and dbcAMP-treated oocytes were at anaphase I or beyond, while the majority of oocytes treated with 8-bromo-cAMP or forskolin had only progressed to metaphase I. Prolonged cultures for 44 h revealed no significant difference in the proportion of treated oocytes reaching anaphase I. to metaphase II compared with controls. IBMX inhibited GVBD in a dose-dependent manner following 24-h cultrue, and at low concentrations its effects was similar to that observed for high concentration of 8-bromo-cAMP and forskolin. The timing of observation is thus crucial for interpreting the effects of these compounds on bovine oocyte maturation. The results suggest that elevation of intracellular cAMP induces a transitory inhibition of bovine oocyte GVBD. Although the bovine oocyte plasma membrane contains adenylate cyclase, it may also possess an active phosphodiesterase that could prevent dbcAMP and forskolin from raising intracellular levels of cAMP suffeciently to maintain meiotic arres for any considerable time. As 8-bromo-cAMP is only partially hydrolyzed by phosphodiesterase, there may be more of this analogue available to stimulate cAMP-dependent protein kinase, which could account for the more pronounced effect observed with 8-bromo-cAMP compared with dbc-AMP.