Morphological effects of caffeine, okadaic acid and genistein in one-cell mouse embryos blocked in G2 by X-irradiation.

Abstract

One-cell mouse embryos of the Balb/c strain normally divide at 18.5 h p.c. (post conception), but they suffer an extremely long G2 arrest when irradiated with 2 Gy X-rays 8 h p.c. at the early pronuclear stage. This could be an indirect effect of radiation on tyrosine dephosphorylation of the p34cdc2 subunit of a maturation or mitosis promoting factor (MPF), which normally occurs at the end of G2. This, in turn, would maintain MPF in an inactivated form and block entry into mitosis. Preliminary studies were undertaken at the morphological level to assess indirectly the validity of this hypothesis. For this purpose, irradiated and control embryos were exposed to different compounds, which are known to interfere, directly or indirectly, with the state of phosphorylation/dephosphorylation of p34cdc2. Caffeine (CAF; 2 mM) did not affect the time of first division of control embryos, but it completely suppressed the radiation-induced G2 arrest of embryos exposed to this compound from 17 h p.c., i.e. 1.5 h before the normal time of first cleavage. Under the same conditions, okadaic acid (OA; 3 microM), a specific inhibitor of phosphatases I and IIA, induced a rapid pronuclear membrane breakdown and a block of all control and irradiated embryos at metaphase. Genistein (GEN; 92 or 185 microM). A potent inhibitor of tyrosine kinases, increased the radiation-induced G2 arrest and even induced a dose-dependent G2 arrest in the control embryos. Embryos were exposed at different times following irradiation to a mixture of either CAF (2 or 5 mM) or OA (3 or 10 microM), and cycloheximide (CH; 5 micrograms/ml), a potent protein synthesis inhibitor. Reversion of G2-arrest by CAF was still seen in embryos exposed to CAF+CH from 17 h p.c. However, the proportion of irradiated embryos eventually able to cleave was lower than that obtained under the conditions of exposure to CAF alone. Embryos exposed to CAF+CH before 17 h p.c. were not able to cleave, regardless of the concentration of CAF used. Nuclear envelope breakdown still occurred in 100% control and irradiated embryos, following exposure to 3 microM OA+CH from 10 h p.c., or to 10 microM OA+CH from 8.5 p.c.(ABSTRACT TRUNCATED AT 400 WORDS)