Fully grown, meiotically immature mouse oocytes were isolated and cultured under varying conditions with the aim of determining a) whether the inhibitory effects of testosterone on oocyte meiotic maturation require the synthesis of new oocyte proteins and b) if the meiosis-inhibiting effects of testosterone and dibutyryl cyclic AMP (dbcAMP) are distinct and can be differentiated. We found that the inclusion of puromycin in culture medium containing testosterone has no effect on the meiosis-inhibiting potency of testosterone or upon the reversibility of testosterone effects. We conclude that testosterone inhibits oocyte meiosis by a mechanism that is independent of protein synthesis. We also found that oocytes exposed to testosterone recover more rapidly, as evidenced by the timing of germinal vesicle breakdown (GVBD) following placement in a control medium, than do oocytes exposed to dbcAMP. Through further investigation of this phenomenon we have determined the sequence of testosterone and dbcAMP effects relative to the time course of GVBD. A testosterone-sensitive event occurs 20 min prior to GVBD, while the dbcAMP-sensitive event precedes GVBD by 41 min. The nature of this difference may involve the differential interaction of testosterone and dbcAMP with a set of puromycin-sensitive proteins that are required for GVBD. When oocytes were initially cultured in medium containing both puromycin and either testosterone or dbcAMP and then moved to medium containing puromycin alone the incidence of GVBD was reduced relative to oocytes never exposed to puromycin. This observation suggests that mouse oocytes contain proteins that are required for GVBD and that experience a high turnover rate. The degree of reduction in GVBD was a function of the length of puromycin exposure and was significantly greater in dbcAMP- than in testosterone-exposed oocytes. If oocytes were initially cultured in medium containing puromycin and dbcAMP, the rate of GVBD upon removal of dbcAMP was initially slow but increased with time. This observation is consistent with the hypothesis that dbcAMP inhibits oocytes at a point prior to the functioning of the puromycin-sensitive proteins. However, if oocytes were cultured in medium containing puromycin and testosterone the rate of GVBD following testosterone removal was not significantly reduced relative to oocytes that were not exposed to puromycin. This observation suggests that testosterone acts to inhibit meiosis at a site beyond the function of the puromycin-sensitive proteins or that testosterone causes a reduction in the turnover rate of these proteins.(ABSTRACT TRUNCATED AT 400 WORDS)