Phosphatidylinositol 3-Kinase Activity Is Critical for Glucose Metabolism and Embryo Survival in Murine Blastocysts

Abstract

The phosphatidylinositol 3-kinase (PI3K) signal transduction pathway is a well known mediator of cell growth, proliferation, and survival signals. Whereas the expression and function of this pathway has been documented during mammalian development, evidence demonstrating the physiologic importance of this pathway in murine preimplantation embryos is beginning to emerge. This study demonstrates that inhibition of the PI3K pathway leads to the induction of apoptosis in both murine blastocysts and trophoblast stem cells. The apoptosis induced in both model systems correlates with a decrease in the expression of the glucose transporter GLUT1 at the plasma membrane. In addition, blastocysts cultured in the presence of the PI3K inhibitor LY-294002 display a decrease in both 2-deoxyglucose uptake and hexokinase activity as compared with control blastocysts. To determine the impact of PI3K inhibition on pregnancy outcome, embryo transfer experiments were performed. Blastocysts cultured in the presence of LY-294002 demonstrate a dramatic increase in fetal resorptions as compared with control embryos. Finally, we demonstrate that impairment of glucose metabolism via iodoacetate, a glyceraldehyde-3-phosphate dehydrogenase inhibitor, is sufficient to induce apoptosis in both blastocysts and trophoblast stem cells. Moreover, blastocysts treated with iodoacetate result in poor pregnancy outcome as determined by embryo transfer experiments. Taken together these data demonstrate the critical importance of the PI3K pathway in preimplantation embryo survival and pregnancy outcome and further emphasize the importance of glucose utilization and metabolism in cell survival pathways.The blastocyst stage of murine preimplantation development occurs approximately 4 days post-fertilization. At this stage of embryonic development the first cell differentiation step has occurred. The blastocyst is comprised of the epithelial trophectoderm, which is the layer of cells that develop into the placenta, and the inner cell mass, which consists of the pluripotent cells that gives rise to the embryo proper. Prior to implantation, the developing embryo is dependent on signals generated by growth factors that are either made by the embryo itself or are present in the maternal environment. These growth factors are known to regulate cellular proliferation and differentiation during mammalian preimplantation development (1, 2). Importantly, the preimplantation embryo expresses a number of growth factor receptors known to activate the phosphatidylinositol 3-kinase (PI3K) 2 pathway including the insulin and insulin-like growth factor-I receptors (3, 4).PI3K is a lipid kinase that phosphorylates the D-3 position on the inositol ring in phosphoinositides (5-7). It is a heterodimeric enzyme that consists of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit. A number of extracellular signals activate PI3K including insulin and other growth factors. Insulin and insulin-like growth factor-I have been shown to have both mitogenic and anti-apoptotic ...

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

“…Similar to what was previously found in lymphocytes deprived of exogenous growth factors (Whetton et al 1984, Kan et al 1994, Rathmell et al 2000, Vander Heiden et al 2001 inhibition of the PI3-K/Akt pathway in murine blastocysts using LY-294002, resulted in decreased cell surface expression of GLUT1 with a corresponding decrease in 2-deoxyglucose uptake (Fig. 4) (Riley et al 2005a). Along with the decrease in glucose utilization, the blastocysts displayed increased levels of apoptosis.…”

“…We have also cultured in vivo derived blastocyst for 30 h in vitro, in the presence of increasing concentrations of LY-294002 and demonstrated a dose-dependent increase in the number of TUNEL-positive nuclei per embryo as compared with controls (Fig. 3A) (Riley et al 2005a). A final study conducted by Kawamura et al (2005) demonstrated that TGF-a has anti-apoptotic effects on murine preimplantation embryos exposed to suboptimal culture conditions.…”

The impact of obesity on egg quality

“…The effect of insulin treatment on other enzymes in the insulin signaling pathway in oocytes or cumulus cells is unknown [102]. The IGF-1 knockout mice have decreased granulosa cell proliferation and decreased GLUT1 [89,104], similar to results observed in blastocysts following PI3K inhibition [98]. Feeding mice a high fat diet for 16 weeks leads to weight gain and hyperinsulinemia, poor oocyte quality, and impaired ovulation [105].…”

“…It is present and functional in the preimplantation embryo [97] and studies conducted using embryos may shed light on what could be happening in the oocyte or cumulus cells where fewer studies have been conducted. Insulin signaling through the PI3K pathway mediates insulin stimulated glucose uptake in the blastocyst stage embryo, and inhibition of this pathway blocks insulin stimulated glucose uptake and increases apoptosis [98]. Elevated insulin or IGF-1 in-vitro can reduce insulin stimulated glucose uptake and lead to apoptosis in mouse blastocysts [99,100].…”

Essential role of the CUL4B ubiquitin ligase in extra-embryonic tissue development during mouse embryogenesis

“…Fresh deciduas were fixed in 4% paraformaldehyde overnight at 4 °C, dehydrated and embedded in paraffin. Whole mount IF on blastocysts was performed as previously described [39]. Standard IF was performed on PFA-fixed paraffin sections (6 µm) (E5.0) or frozen sections (E7.5) following procedures as previously described [40].…”