Interphase-like chromatin configuration induced by cycloheximide in maturing pig oocytes: effects of protein phosphatase inhibitors.

Abstract

Embryo cloning methods could greatly benefit from the manipulation of cell cycle in oocytes from large domestic mammals. The present study was undertaken to examine the effects of the protein synthesis inhibitor cycloheximide and the inhibitors of protein phosphatases 1 and 2A okadaic acid and calyculin A on maturing pig oocytes. Cycloheximide treatment (10 micrograms/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 percentage reached its plateau after 12 h of cycloheximide treatment. ICC is fully reversible. The addition of okadaic acid (0.5 microM) inhibited the ICC in cycloheximide-treated maturing oocytes and allowed the completion of maturation in 55% of them. In oocytes with ICC, the immunocytochemistry for tubulin revealed the rearrangement of microtubule into an interphase meshwork and these oocytes lost their ability to induce tubulin assembly, as shown after short-time taxol treatment. The addition of okadaic acid prevented this microtubule rearrangement and preserved a certain level of tubulin assembly. Calyculin appeared to be more effective than okadaic acid in the prevention of ICC. It is concluded that de novo protein synthesis is necessary during a certain period of meiotic maturation for the maintenance of metaphase chromatin configuration in pig oocytes. This protein (or proteins) acts through the inhibition of endogenous protein phosphatases, probably protein phosphatase of 2A type.