Phosphate exposure during the late 1-cell and early 2-cell stages induces a time-specific decrease in cyclin B and cdc25B mRNAs in AKR/N mouse embryos in vitro.

Abstract

Phosphate induces 2-cell block in AKR/N mouse embryos in vitro. In an attempt to define the mechanism responsible for the inhibitory effect of phosphate, the critical period for this effect was determined. Then the amounts of the mRNAs for cyclin B and cdc25B, factors related to activation of M-phase promoting factor (MPF), were measured by quantitative reverse transcription-polymerase chain reaction. Exposure to phosphate during the late 1-cell (10-20 h after insemination) and early 2-cell stages (0-12 h after first cleavage) was found to induce 2-cell block in AKR/N mouse embryos. This period corresponds to the initiation of zygotic gene activation (ZGA). The presence of phosphate during second cleavage had no effect on 2-cell block of the embryos. The relative levels of cyclin B and cdc25B mRNAs did not change significantly during the 1-cell stage and decreased during the early 2-cell stage to almost half of the initial levels. When the amounts of mRNA in embryos cultured with and without phosphate were compared they were found to be almost identical even in the 2-cell block embryos, and a significant decrease in mRNA was observed only 33 h after insemination in embryos about to undergo phosphate block at the 2-cell stage. These results show that phosphate does not directly inhibit MPF activity and confirm the presence of cyclin B and cdc25B even in 2-cell block embryos. Furthermore, the fact that the decrease in mRNA levels corresponded to the critical period for the inhibitory effect of phosphate suggests that suppression of initial ZGA induction is involved in the 2-cell block of mouse embryos in vitro.