Role of calmodulin in blastocyst formation in the mouse

Preimplantation mouse embryos were recovered by flushing the oviducts on Day 3 at 09:30-10:00 h, 15:30-16:00 h and 21:30-22:00 h: When placed in culture for 48 h, 79% of the 4-8 cell embryos recovered at 09:30-10:00 h developed into blastocysts, but a large number of these embryos failed to form blastocysts when exposed to trifluoperazine, a calmodulin antagonist, at 0.5 or 0.6 microM in culture. About 45% of the embryos recovered at 15:30-16:00 h were compacted and blastocyst formation was again markedly depressed in the presence of the drug. Advanced compacted embryos recovered at 21:30-22:00 h showed normal development into blastocysts in the presence of 0.6 microM-trifluoperazine. Trifluoperazine sulphoxide (the inactive form of trifluoperazine) at 0.6 or 1.2 microM concentration had no effect on blastocyst formation of uncompacted embryos recovered at 09:30-10:00 h. These embryos and those recovered at 21:30-22:00 h and developed into blastocysts in the presence of trifluoperazine were transferred to Day-4 pseudopregnant mice and healthy young were born. When exposed to calcium-free medium or medium containing trifluoperazine all compacted embryos recovered at 18:30 h became decompacted; development to the blastocyst stage was normal in medium alone but reduced when trifluoperazine was added. Compacted embryos recovered at 23:00 h showed 100% decompaction in the calcium-free medium but completely failed to decompact in the presence of 0.6 microM-trifluoperazine. We suggest that extracellular calcium is essential for the continuance of compaction, while intracellular calcium is required only during the initial phase of this process.