Characterization and functional significance of calcium transients in the 2-cell mouse embryo induced by an autocrine growth factor

Growth of preimplantation embryos is influenced by autocrine trophic factor s that need to act by the 2-cell stage, but their mode of action is not yet described, This report shows that late zygote and 2-cell stage mouse embry os responded to embryo derived platelet-activating factor (PAF) with transi ent increases in intracellular calcium concentration ([Ca2+](i)). [Ca2+](i) transients were single global events and were specifically induced by embr yo-derived PAF. They were blocked by inhibition of phospholipase C (U 73122 ) and an inositol trisphosphate (IP3) receptor antagonist (xestospongin C), indicating the release of calcium from IP3-sensitive intracellular stores. Transients were also inhibited by the absence of calcium from extracellula r medium and partially inhibited by treatment with dihydropyridine (nifedip ine, 10 mu M), but not pimozide (an inhibitor of an embryonic T-type calciu m channel). (+/-)BAY K8644 (an L-type channel agonist) induced [Ca2+](i) tr ansients, yet these were completely inhibited by nifedipine (10 mu M). The complete inhibition of BAY K8644, but only partial inhibition of PAF by nif edipine shows that L-type channels were only partly responsible for the cal cium influx. Depolarization of 2-cell embryos by 50 mM K+ did not inhibit P AF-induced calcium transients, showing that the influx channels were not vo ltage-dependent. Depletion of intracellular calcium stores by thapsigargin revealed the presence of store-operated channels. The interdependent requir ement for IP3-sensitive internal calcium stores and extracellular calcium i n the generation of PAF-induced transients may be explained by a requiremen t for capacitative calcium entry via store-operated channels. A functionall y important role for the PAF-induced transients is supported by the observa tion that inhibition of [Ca2+](i) transients by a PAF-antagonist (WEB 2086) or an intracellular calcium chelator (1,2-bis (2-aminophenoxy)-ethane-N,N, N',N'-tetraacetic acid tetrakis-acetoxymethyl ester; BAPTA-AM) caused marke d inhibition of early embryo development. Growth inhibition by BAPTA-AM was relieved by addition of exogenous PAF.