Effect of the oxidative phosphorylation uncoupler 2,4-dinitrophenol on hypoxia-inducible factor-regulated gene expression in bovine blastocysts

Abstract

In cattle embryos, development to the blastocyst stage is improved in the presence of 10 micro;m 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, coincident with an increase in glycolytic activity following embryonic genome activation. The present study examined redox-sensitive gene expression and embryo development in response to the addition of DNP post-compaction. 2,4-Dinitrophenol increased the expression of hypoxia-inducible factor 1alpha and 2alpha (HIF1alpha, HIF2alpha) mRNA. Although HIF1alpha protein remained undetectable in bovine blastocysts, HIF2alpha protein was localised within the nucleus of trophectoderm and inner cell mass (ICM) cells of blastocysts cultured in the presence or absence of DNP, with a slight increase in staining evident within the ICM in blastocysts cultured in the presence of DNP. However, the expression of GLUT1 and VEGF mRNA, genes known to be regulated by HIFs, was unaffected by the addition of DNP to the culture. Although the development of Grade 1 and 2 blastocysts was unaltered by the addition of DNP post compaction in the present study, a significant increase in the proportion of ICM cells was observed. Results indicate that 10 microm DNP improves the quality of bovine embryos, coincident with increased HIF2alpha protein localisation within ICM cells and increased HIFalpha mRNA levels. Therefore, the results demonstrate redox-regulated expression of HIF2.

Prolyl‐hydroxylase inhibition and HIF activation in osteoblasts promotes an adipocytic phenotype

“…Previous studies have demonstrated that mRNA and protein levels of HIFs are not necessarily directly related. For example, uncoupling of oxidative phosphorylation in blastocysts leads to an increase in mRNA levels of both HIF-1a and HIF-2a mRNA levels, but only an increase in HIF-2a protein levels is observed [Harvey et al, 2004]. Consistent with our findings, others have found that HIF-2a protein levels are upregulated in MG63 osteoblast-like cells within 3 h and at 24 h of hypoxic exposure , while HIF-1a protein levels were not induced under these conditions (although they could be induced by treatment with a proteosome inhibitor) .…”

Blastocyst metabolism

“…Species-specific responses to reduced oxygen culture may reflect differences in the mode of implantation, or interactions between culture medium and oxygen concentration that alter cellular redox status and thereby metabolic activity (Harvey et al 2002). Indeed, modulation of oxidative phosphorylation with the uncoupler 2,4-dinitrophenol during postcompaction bovine embryo culture increases the expression of HIF1a and HIF2a mRNA and modulates blastocyst cell allocation (Harvey et al 2004b). These differences reinforce the need to establish appropriate culture conditions and oxygen levels for each species.…”

The embryonic stress response to in vitro culture: insight from genomic analysis

“…By affecting oxidative homeostasis, it might actually decrease energy production by the mitochondria (Harvey et al 2004a). Uncouplers and inhibitors of OXPHOS appear to affect embryo development in a similar manner (Thompson et al 2000, Machaty et al 2001 as well as the expression of redox-sensitive genes such as HIF1A and HIF2A (Harvey et al 2004b). Reduced oxygen tension increases the expression of metabolic genes involved in glycolysis such as LDHA (Harvey et al 2007).…”