CHANGES IN MATURATION-PROMOTING ACTIVITY IN THE CYTOPLASM OF PIG OOCYTES THROUGHOUT MATURATION

Maturation-promoting factor (MPF) was examined in maturing pig oocytes by electrofusing them with germinal vesicle (GV) oocytes. Oocytes containing high levels of MPF (MI or MII stages) induced the breakdown of the GV introduced by fusion and the formation of the metaphase plate in 1 hr. A similar effect was seen when two or three GV oocytes were fused with a MII oocyte and then incubated for 1 hr in the presence of cycloheximide (a specific protein synthesis inhibitor), indicating that high levels of preformed MPF are present at the metaphase stage. During the maturation in vitro of cumulus-enclosed oocytes, a first sharp rise in MPF was seen between 26 and 29 hr of culture (MI stage); MPF declined after 2 hr (AI-TI stages) and again reached high levels at 35 hr, where it remained for the rest of maturation. Denuded oocytes showed a similar behavior, but MPF appeared 9 hr earlier and the rise, due to the asynchronous maturation of these oocytes, was not as sharp as in cumulus enclosed oocytes. Cycloheximide was used to study protein synthesis requirements for oocyte maturation. Intact GV were observed after 44 hr of culture when cycloheximide was added at 26 hr or earlier, and chromosome decondensation and pronuclear formation were observed when the drug was added at 32 hr. Transcriptional requirements were investigated by treating the oocytes with alpha-amanitin, an RNA polymerase inhibitor. This drug could completely inhibit the maturation of cumulus-enclosed oocytes, but this was a somatic cell-mediated effect since denuded oocytes were insensitive to this treatment. Enucleated oocytes exhibited an increase in MPF after 24 hr of culture, and low levels of this factor were seen after 40 h of maturation. These data indicate that both rises in MPF require active protein synthesis, whereas transcription is not necessary for the resumption of meiotic cycle. Nuclear activity may be required for the second rise in MPF.