title: Characterization and functional significance of calcium transients in the 2-cell mouse embryo induced by an autocrine growth factor.
abstract: Growth of preimplantation embryos is influenced by autocrine trophic factors 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 embryos responded to embryo-derived platelet-activating factor (PAF) with transient increases in intracellular calcium concentration ([Ca(2+)](i)). [Ca(2+)](i) transients were single global events and were specifically induced by embryo-derived PAF. They were blocked by inhibition of phospholipase C (U 73122) and an inositol trisphosphate (IP(3)) receptor antagonist (xestospongin C), indicating the release of calcium from IP(3)-sensitive intracellular stores. Transients were also inhibited by the absence of calcium from extracellular medium and partially inhibited by treatment with dihydropyridine (nifedipine, 10 micrometer), but not pimozide (an inhibitor of an embryonic T-type calcium channel). (+/-)BAY K8644 (an L-type channel agonist) induced [Ca(2+)](i) transients, yet these were completely inhibited by nifedipine (10 micrometer). The complete inhibition of BAY K8644, but only partial inhibition of PAF by nifedipine shows that L-type channels were only partly responsible for the calcium influx. Depolarization of 2-cell embryos by 50 mm K(+) did not inhibit PAF-induced calcium transients, showing that the influx channels were not voltage-dependent. Depletion of intracellular calcium stores by thapsigargin revealed the presence of store-operated channels. The interdependent requirement for IP(3)-sensitive internal calcium stores and extracellular calcium in the generation of PAF-induced transients may be explained by a requirement for capacitative calcium entry via store-operated channels. A functionally important role for the PAF-induced transients is supported by the observation that inhibition of [Ca(2+)](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 marked inhibition of early embryo development. Growth inhibition by BAPTA-AM was relieved by addition of exogenous PAF.
authors.affiliation: Human Reproduction Unit, Department of Physiology, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia.
authors.forename: M
authors.initials: M
authors.lastname: Emerson
authors.forename: A R
authors.initials: AR
authors.lastname: Travis
authors.forename: R
authors.initials: R
authors.lastname: Bathgate
authors.forename: T
authors.initials: T
authors.lastname: Stojanov
authors.forename: D I
authors.initials: DI
authors.lastname: Cook
authors.forename: E
authors.initials: E
authors.lastname: Harding
authors.forename: D P
authors.initials: DP
authors.lastname: Lu
authors.forename: C
authors.initials: C
authors.lastname: O'Neill
mesh_terms: D000818:Animals; D019898:Autocrine Communication; D001755:Blastocyst / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D004963:Estrenes / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000037:antagonists & inhibitors / Q000378:metabolism; D047028:Macrocyclic Compounds; D051379:Mice; D010080:Oxazoles / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010972:Platelet Activating Factor / Q000378:metabolism*; D011760:Pyrrolidinones / Q000494:pharmacology
chemical_list: D004963:Estrenes; D047028:Macrocyclic Compounds; D010080:Oxazoles; D010726:Phosphodiesterase Inhibitors; D010972:Platelet Activating Factor; D011760:Pyrrolidinones; C108644:xestospongin A; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium