Direct evidence for the action of phosphatidylinositol (3,4,5)-trisphosphate-mediated signal transduction in the 2-cell mouse embryo.
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 (PI3kinase), such as wortmannin and LY 294002, blocked these calcium transients, implicating the generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in autocrine signal transduction in the early embryo. Perfusion of the embryo cytoplasm with a blocking antibody to PIP3 inhibited paf-induced calcium transients and hyperpolarization of the membrane potential. Furthermore, direct infusion of PIP3 into the embryo induced a nifedipine (10 micromol/L)- and diltiazem (10 micromol/L)-sensitive calcium current in the 2-cell embryo. PIP3 acts as a docking site on membranes for proteins that contain pleckstrin homology domains, such as the 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 the expression of both AKT and phospholipase C gamma 1 by immunolocalization. Paf induced increased accumulation of serine 473-phosphorylated AKT in the region of the plasma membrane, consistent with its recruitment to membrane PIP3. Inhibitors of PI3kinase, such as LY294002, and of AKT, e.g., deguelin and AKT-inhibitor, reduced zygote development in a dose-dependent manner, and this inhibition was partially reversed by the addition of paf to the culture medium. These results provide the first direct evidence that PIP3 and its responsive signaling pathways act in the 2-cell embryo. Since signal transduction via PI3kinase has important roles in governing the cell survival pathways, these results support the hypothesis that autocrine embryotropins, such as paf, act as survival factors.
Li
Yan
Y
Disciplines of Medicine and Physiology, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia.
Chandrakanthan
Vashe
V
Day
Margot L
ML
O'Neill
Chris
C
D000818:Animals; D001755:Blastocyst / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008817:Mice, Mutant Strains; D009025:Morpholines / Q000494:pharmacology; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism* / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051966:Phospholipase C gamma / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D010972:Platelet Activating Factor / Q000378:metabolism* / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D012402:Rotenone / Q000031:analogs & derivatives / Q000494:pharmacology; D015398:Signal Transduction; D015053:Zygote / Q000187:drug effects / Q000378:metabolism*
D002867:Chromones; D009025:Morpholines; D018129:Phosphatidylinositol Phosphates; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010972:Platelet Activating Factor; D012333:RNA, Messenger; C060974:phosphatidylinositol 3,4,5-triphosphate; D012402:Rotenone; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D051057:Proto-Oncogene Proteins c-akt; D051966:Phospholipase C gamma; C107676:deguelin; D002118:Calcium