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

Abstract

The growth and survival of the preimplantation mammalian embryo may be regulated by several autocrine trophic factors that have redundant or overlapping actions. One of the earliest trophic factors to be produced is embryo-derived platelet-activating factor (1-O-alky-2-acetyl-sn-glyceryl-3-phosphocholine). The addition of platelet-activating factor to embryo culture media exerted a trophic effect, but structurally related lipids (3-O-alky-2-acetyl-sn-glyceryl-1-phosphocholine, 1-O-alky-sn-glyceryl-3-phosphocholine, octadecyl-phosphocholine) had no effect. Platelet-activating factor induced a pertussis toxin-sensitive [Ca2+]i transient in two-cell embryos that did not occur in platelet-activating factor-receptor null (Pafr–/–) genotype embryos. Fewer Pafr–/– mouse zygotes developed to the blastocyst stage in vitro compared with Pafr+/+ zygotes (P<0.02), those that developed to blastocysts had fewer cells (P<0.001) and more cells with fragmented nuclei (P<0.001). The inhibition of 1-O-phosphatidylinositol 3-kinase (LY294002 (3 μM and 15 μM) and wortmannin (10 nM and 50 nM)) caused a dose-dependent inhibition of platelet-activating factor-induced [Ca2+]i transients (P<0.001). The two-cell embryo expressed 1-O-phosphatidylinositol 3-kinase catalytic subunits p110α, β, γ and δ, and regulatory subunits p85α and β. LY294002 and wortmannin each caused a significant reduction in the proportion of embryos developing to the morula and blastocyst stages in vitro, reduced the number of cells within each blastocyst, and significantly increased the proportion of cells in blastocysts with fragmented nuclei. The results indicate that embryo-derived platelet-activating factor (and other embryotrophic factors) act through its membrane receptor to enhance embryo survival through a 1-O-phosphatidylinositol 3-kinase-dependent survival pathway.

TCL1 promotes blastomere proliferation through nuclear transfer, but not direct phosphorylation, of AKT/PKB in early mouse embryos

“…This conclusion is in agreement with previous observations that mouse preimplantation embryo development requires PI3K activity from the 8/16-cell stage. 8 If TCL1 is not relevant to AKT phosphorylation in one-cell and two-cell embryos, is this factor needed for the phosphorylated AKT transfer to nucleus? We have addressed this issue by determining the intracellular distribution of Ser473/ Thr308-phosphorylated AKT in two-cell embryos that were genetically deficient in TCL1.…”

“…Embryo exposure to either 10-20 mM LY294002 or 6-24 nM Wortmannin negatively affected preimplantation development from the four-cell stage (Figure 1b), in agreement with previous observations. 8 Interestingly, both inhibitors had no apparent effect on the occurrence of the first embryo cleavage, even when the inhibitor concentration in the medium was raised to extremely high values as 100 mM LY294002 or 100 nM Wortmannin (not shown). Embryos exposed to such high inhibitor concentration displayed an altered overall morphology and did not progress beyond the four-cell stage, indicating that the inhibitor had effectively damaged cellular functions (Supplementary Figure S2a).…”

Glucose utilization and the PI3-K pathway: mechanisms for cell survival in preimplantation embryos

“…Murine 2-cell embryos were shown to express the mRNA of multiple PI3-K isoforms including p85a and b and p110 a, b, g, d (Lu et al 2004). In addition, Kawamura et al (2005) demonstrated the presence of p110 transcripts from the oocyte through the hatched blastocysts stage of murine preimplantation development.…”

“…Lu et al (2004) demonstrated the importance of the PI3-K pathway in mammalian preimplantation embryo development by showing that the activation of PI3-K by an embryonic trophic factor, platelet-activating factor (PAF), is critical for embryo development and survival. PAF treatment of 2-cell embryos results in a transient increase in calcium that is inhibited by both LY-294002 and wortmannin implying that PI3-K activity is required for this PAF-induced biological response.…”

“…In addition, a separate global gene expression study demonstrated that genes involved in inositol phosphate and calcium signaling such as Pik3c2a (a class II PI3-K a polypeptide) are increased in activated as compared with dormant blastocysts (Hamatani et al 2004). The importance of the PI3-K pathway during the preimplantation period was highlighted by a study that demonstrated that inhibition of PI3-K results in decreased numbers of embryos that develop to the morula and blastocyst stage in vitro when cultured from a zygote stage, similar to Pafr deficient embryos (Lu et al 2004). The blastocysts that did develop contained fewer cells and a larger number of fragmented nuclei.…”