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Fig. 4. PI3K activity promotes early embryo development and survival. (A) Chronic PI3K inhibition with LY294002 prevents development beyond the two-cell stage. One-cell embryos were cultured continuously in the presence of LY294002 (10 µM, grey) or the equivalent amount of DMSO (black), in 3 ml of medium. Over 100 embryos were scored in each experimental condition. ^*P<0.0001; ^#P<0.01. (B) Developmental competence of embryos injected with 0.18 µg/µl (grey bar, 1x) or 1.8 µg/µl (dark green, 10x) of GFP-PH_GRP1 cRNA, or GFP cRNA (2.0 µg/µl, light green). Control, non-injected embryos are shown in black. Culture was performed in high-density conditions. The number of embryos examined is indicated on the graph. ^*P<0.0001. (C) PI3K inhibition with LY294002 prevents the morula-blastocyst transition. Embryos were cultured in high-density conditions until they reached a given stage, before being exposed to LY294002 (without mineral oil). Embryos were checked the next day to examine their progression to the next developmental stage (indicated on the x-axis). Thirty to 50 embryos were scored for each developmental transition. ^*P<0.0001. (D) 16-32 cell morula recovered from a high-density culture and exposed to 10 µM LY294002 for 20 hours, without mineral oil. TUNEL staining reveals apoptosis in virtually all blastomeres (right panel). The arrowhead indicates a small blastocoel-like cavity. (E) Expanded blastocyst obtained after culturing a 16-32 cell morula in the presence of DMSO for 20 hours, without mineral oil. Little apoptosis was detectable (TUNEL staining, right). (F) Eight-cell embryo cultured in the presence of ECCD-1 from the one-cell stage, in high-density conditions. All blastomeres were negative for TUNEL staining (right). (G) 16-32 cell embryo cultured in the presence of ECCD-1 from the one-cell stage, in high-density conditions, exhibiting extensive TUNEL staining (right). All TUNEL images (D-G) are projections of multiple confocal sections across the whole embryo, and are representative of 12-26 similar observations.

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