Embryo

Direct Evidence for the Action of Phosphatidylinositol (3,4,5)-Trisphosphate-Mediated Signal Transduction in the 2-Cell Mouse Embryo

Abstract
Paf (1-o-alkyl-2-acetyl-sn-gylcero-3-phosphocholine) is a putative autocrine survival factor for the preimplantation embryo. It acts to induce receptor-mediated calcium transients in the early embryo. Inhibitors of 1-o-phosphatidylinositol-3-kinase (wortmannin and LY 294002) blocked these calcium transients, implicating the generation of phosphatidylinositol (3,4,5)-trisphosphate in autocrine signal transduction in the early embryo. Perfusion of the embryo's cytoplasm with a blocking antibody to phosphatidylinositol (3,4,5)-trisphosphate inhibited paf-induced calcium transients and hyperpolarization of membrane potential. Furthermore, direct infusion of phosphatidylinositol (3,4,5)-trisphosphate into the embryo induced a nifedipine (10 µM) and diltiazem (10 µM) sensitive calcium current in the 2-cell embryo. Phosphatidylinositol (3,4,5)-trisphosphate acts as a docking site on membranes for proteins containing pleckstrin homology domains, such as thymoma viral proto-oncogene protein (AKT) and phospholipase C gamma. The 2-cell embryo expressed three genes for AKT (Akt 1-3) and two genes for phospholipase C gamma (Plcg1 and Plcg2), and we confirmed expression of both AKT and phospholipase C gamma 1 by immunolocalization. Paf induced the increased accumulation of serine 473 phosphorylated AKT in the region of the plasma membrane, consistent with its recruitment to membrane phosphatidylinositol (3,4,5)-trisphosphate. Inhibitors of 1-o-phosphatidylinositol-3-kinase (LY294002) and AKT (deguelin and AKT-inhibitor) reduced zygote development in a dose-dependent manner, and this was partially reversed by the addition of paf to culture media. The results provide the first direct evidence that phosphatidylinositol (3,4,5)-trisphosphate and its responsive signaling pathways act in the 2-cell embryo. Since signal transduction via 1-o-phosphatidylinositol-3-kinase has important roles in governing the cell survival pathways, the results support the hypothesis that autocrine embryotrophins, such as paf, act as survival factors.

Key words: Embryo • Calcium • Developmental biology • Early development

This article has been cited by other articles:

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