INTERPHASE-LIKE CHROMATIN CONFIGURATION INDUCED BY CYCLOHEXIMIDE IN MATURING PIG OOCYTES - EFFECTS OF PROTEIN PHOSPHATASE INHIBITORS

Embryo cloning methods could greatly benefit from the manipulation of cell cycle in oocytes from large domestic mammals. The present study w as undertaken to examine the effects of the protein synthesis inhibito r cycloheximide and the inhibitors of protein phosphatases 1 and 2A ok adaic acid and calyculin A on maturing pig oocytes. Cycloheximide trea tment (10 mu g/ml) induced an interphase-like chromatin configuration (ICC) in maturing oocytes. Up to 69% of the oocytes exhibited ICC when treated with cycloheximide after 24 h of in vitro culture. ICC starts to appear after a 4 h exposure to cycloheximide and the ICC percentag e reached its plateau after 12 h of cycloheximide treatment. ICC is fu lly reversible. The addition of okadaic acid (0.5 mu M) inhibited the ICC in cycloheximide-treated maturing oocytes and allowed the completi on of maturation in 55% of them. In oocytes with ICC, the immunocytoch emistry for tubulin revealed the rearrangement of microtubule into an interphase meshwork and these oocytes lost their ability to induce tub ulin assembly, as shown after short-time taxol treatment. The addition of okadaic acid prevented this microtubule rearrangement and preserve d a certain level of tubulin assembly. Calyculin appeared to be more e ffective than okadaic acid in the prevention of ICC. It is concluded t hat de novo protein synthesis is necessary during a certain period of meiotic maturation for the maintenance of metaphase chromatin configur ation in pig oocytes. This protein (or proteins) acts through the inhi bition of endogenous protein phosphatases, probably protein phosphatas e of 2A type.