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First published online March 12, 2004
doi: 10.1242/10.1242/jcs.00991

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Trophic signals acting via phosphatidylinositol-3 kinase are required for normal pre-implantation mouse embryo development

¹ Human Reproduction Unit, Department of Physiology, University of Sydney, Royal North Shore Hospital, St Leonards, NSW 2065, Australia
² CREST of Japan Science and Technology Corporation, Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
³ Human Reproduction Unit, Royal North Shore Hospital, St Leonards, NSW 2065, Australia

View larger version (12K): [in a new window]   Fig. 1. (A) The effect of pertussis toxin on PAF-induced Ca2+ transients in two-cell embryos. Traces show the mean [Ca2+]i from the number of embryos shown in brackets. The experiments were repeated three times. Embryos were pretreated with the concentration of pertussis indicated and then challenged with 37 nM PAF/ml. (B) The role of the PAF-receptor in PAF-induced calcium transients in two-cell embryos and expression of receptor in human embryos. Two-cell embryos collected from either Pafr+/+ or Pafr–/– parents were exposed to rPAF acetylhydrolase and then challenged with 20 ng PAF/ml. Traces are representative of over 30 embryos from three replicates.

View larger version (34K): [in a new window]   Fig. 2. The effect of Pafr expression and embryo density on the development of zygotes in vitro. Pafr+/+ or Pafr–/– embryos were cultured either individually (1/10) or in groups of 10 (10/10) in 10 µl drops of media for 96 hours. (A) The proportion of embryos achieving expected developmental landmarks at each 24 hour interval was assessed, and (B) for those zygotes that developed to morphologically normal blastocysts after 96 hours culture the number of cells per embryo (mean ± s.d.) with apparently normal or obviously fragmented nuclei was assessed.

View larger version (17K): [in a new window]   Fig. 3. The effect of inhibitors of various signal transduction agents on PAF-induced Ca2+ transients in two-cell embryos. Traces show the mean intracellular calcium of at least 30 embryos from three replicates. Embryos were treated with PAF:acetylhydrolase, then with inhibitors shown for 30 minutes and then challenged with 37 nM PAF/ml in the presence of inhibitor.

View larger version (44K): [in a new window]   Fig. 4. (A) The effect of inhibitors of phosphoinositide 3-kinase on PAF-induced Ca2+ transients in two-cell embryos. The results are the mean and s.e.m. of at least 30 embryos from three replicates. Embryos were treated with PAF:acetylhydrolase, then with the inhibitors and concentrations shown and then challenged with 37 nM PAF/ml in the presence of inhibitors. (B) The expression of phosphoinositide 3-kinase subunits in the two-cell embryo. A: mRNA for P110 catalytic subunits in two-cell embryos and brain tissue. M is molecular weight size markers (GeneRuler DNA Ladder Mix). Lane 1 lacked reverse transcriptase; Lane 2, no template acted as a negative controls; Lane 3, ß-actin (243 bp; positive control); Lane 4, p110 (238 bp); Lane 5, p 110ß (146 bp); Lane 6, p110g (111 bp); and Lane 7, p110 (bp 294). B: mRNA for P85 regulatory subunits in two-cell embryos. M is molecular weight size markers. Lane 1, no reverse transcriptase; Lane 2, no template; Lane 3, two-cell ß-actin; Lane 4, P85 -subunit (128bp), Lane 8 P85, ß-subunit (159 bp).

View larger version (15K): [in a new window]   Fig. 5. The role of phosphoinositide 3-kinase in PAF-induced Ca2+-influx as assessed by Mn2+-quenching of Fura-2. The relative fluorescence (arbitrary units) of Fura-2 loaded two-cell embryos measured at 360 nm in perfusion media in which calcium has been replaced with 0.1 mM Mn2+. The lines in each graph show the mean traces from the number of embryos shown in brackets. The boxed areas on the x-axis represent the time interval that the treatments indicated were applied.

View larger version (30K): [in a new window]   Fig. 6. The effect of the phosphoinositide 3-kinase inhibitor LY294002 on the development of zygotes in vitro. Zygotes were cultured individually in 10 µl drops of media for 96 hours. The three groups of bars in A and B show the results for incubation with 0 µl, 3 µl and 15 µl LY294002, respectively. (A) The proportion of embryos achieving expected developmental landmarks at each 24 hour interval was assessed, and (B) for those zygotes that developed to morphologically normal blastocysts after 96 hours culture the number of cells per embryo (mean ± s.d.) with apparently normal or obviously fragmented nuclei was assessed.

View larger version (30K): [in a new window]   Fig. 7. The effect of the phosphoinositide 3-kinase inhibitor Wortmannin on the development of zygotes in vitro. The zygotes were cultured individually in 10 µl drops of media for 96 hours. (A) The proportion of embryos achieving expected developmental landmarks at each 24 hour interval was assessed, and (B) for those zygotes that developed to morphologically normal blastocysts after 96 hours culture the number of cells per embryo (mean ± s.d.) with apparently normal or obviously fragmented nuclei was assessed.

View larger version (32K): [in a new window]   Fig. 8. The effect on zygotes treated with the reversible phosphoinositide 3-kinase inhibitor LY294002 inhibitor (LY294002) for 30 hours, followed by culturing without LY294002 for a further 66 hours. Zygotes were cultured either individually in 10 µl drops of media for 96 hours. The three groups of bars in A and B show the results for incubation with 0 µl, 3 µl and 15 µl LY294002, respectively. (A) The proportion of embryos achieving expected developmental landmarks at each 24 hour interval was assessed. (B) For those zygotes that developed to morphologically normal blastocysts after a total culturing time of 96 hours, the number of cells with apparently normal and also obviously fragmented nuclei was assessed per embryo (mean ± s.d.).

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