cAMP-responsive element-binding protein expression and regulation in the mouse preimplantation embryo

Gene expression from the new embryonic genome is required for normal preimplantation embryo development. Two members of the cAMP-responsive element-binding protein (Creb) family of transcription factors, Creb1 and activating transcription factor 1 (Atf1), are essential for normal preimplantation development. These transcription factors are activated by phosphorylation. Creb1 mRNA was expressed throughout the preimplantation phase. Cytoplasmic immunolocalization of Creb1 was detected in all preimplantation embryo stages. The antigen was largely excluded from the pronuclei/nuclei at embryonic stages except in the mid-cycle two-cell and compacted eight-cell embryo. Activation-state-specific antibodies showed serine 133 phosphorylated Creb1 localization was similar to Creb1 staining, except that there was no increase in staining at the eight-cell stage. Increased staining of phosphorylated Creb1 was observed in the nucleus of mid-cycle two-cell embryos. Increased expression of phosphorylated Creb1 in the two-cell embryo was induced by brief exposure of embryos to ionomycin, but not by a dibutyryl cAMP. This was blocked by buffering intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester), but not by a cAMP antagonist, Rp-cyclic 3',5'-hydrogen phosphorothioate adenosine. Calmodulin is an intracellular receptor for calcium. Calmodulin mRNA was expressed throughout the preimplantation phase of development. The calmodulin antagonist, W-7, inhibited the ionomycin-induced localization of phosphorylated Creb1 in the nucleus. Treatment of embryos with W-7 caused a dose-dependent inhibition of normal development of zygotes to the blastocysts stage. The study shows Creb1 expression and nuclear localization was dynamically regulated in the early embryo. The marked nuclear accumulation and phosphorylation of Creb1 at the two-cell stage occurred at the time of transcription from the embryonic genome and was regulated in a calcium- and calmodulin-dependent manner.