Figure 1
Inhibition of early events of Xenopus egg fertilization by LY294002, a potent PI 3-kinase inhibitor. (A) Xenopus unfertilized eggs were microinjected with DMSO alone (lanes 1 and 2), 10 μM PP2 (lane 3), 10 μM PP3 (lane 4), 10 μM U73122 (lane 5), or 10 μM LY294002 (lane 6), and subjected to no treatment (lane 1, - sperm) or insemination for 5 min (lanes 2-6, + sperm). SDS (0.1%)-solubilized egg membrane fractions (500 μg/lane) abundant in Src and PLCγ were analyzed for tyrosine phosphorylation of Src (top panel, IB: phospho-Src), activation of Src (middle panels, IVKA: phospho-Cdk1; IVKA: protein amounts of the immunoprecipitated Src), and tyrosine phosphorylation of PLCγ (bottom panel, IB: phospho-PLCγ) as described in "Methods". Asterisks in each panel indicate the positions of the protein bands of interest. (B) Shown are representative traces of sperm-induced Ca2+ release, as monitored by the fluorescent ratio signal, in Xenopus eggs that were co-injected with fura-2 and LY294002 (0 or 10 μM) and inseminated as described in "Methods". (C) Triton X-100-solubilized extracts (100 μg/lane) were prepared from Xenopus eggs that were injected with the indicated inhibitors (lanes 3-5, each 10 μM) and inseminated for 40 min. Extracts from DMSO (0.2%)-injected unfertilized (lane 1) or fertilized eggs (lane 2) were also prepared as controls. Samples were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against phospho-MAPK (top panel), cyclin B2 (middle panel), or Mos (bottom panel) as described in "Methods". (D) The occurrence of first embryonic cleavage was evaluated in fertilized Xenopus embryos that were injected with the indicated concentrations of LY294002 and inseminated for 100 min. Values indicated on each bar are the number of cleaved embryos per number of embryos tested.