Exposure of an embryo to suboptimal environments, including poor embryo culture media or inadequate maternal diet, can disrupt fetal and placental development and whilst the exact mechanisms responsible remain unknown, perturbed embryo metabolism has been implicated. We propose that stress applied to an early embryo causes mitochondrial dysfunction, resulting in a permanent epigenetic change. Thus the aim of this study was to determine the affect of directly perturbing mitochondria in the embryo, on development, metabolism and expression of the ATP-dependant chromatin remodelling protein, ATRX. Zygotes collected from gonadotrophin stimulated C57BL/6xCBA mice were cultured to the two-cell stage and then exposed to one of three treatments; control medium (C), medium lacking pyruvate (-P; embryos dependant on the mitochondrial Malate Aspartate Shuttle, MAS) or medium lacking pyruvate plus 5µM amino-oxyacetate (AOA), a specific MAS inhibitor (-P+AOA). Blastocyst development and metabolism were assessed by determining cell number and allocation, glycolysis, and ATP:ADP ratio. Relative gene expression of ATRX, was examined using RT PCR. Embryos dependant on the MAS alone (-P) had significantly decreased blastocyst development (87.1% v. 98.2%, P < 0.05), with a compensatory increase in glycolysis (0.20 v. 0.07 pmol/cell/hr, P < 0.001) despite a decrease in ATP:ADP (0.10 v. 0.13, P < 0.06), relative to the control. Inhibition of the MAS (-P+AOA) further reduced blastocyst development,(77.3%, P < 0.001) and decreased ATP:ADP (0.08, P < 0.004), but there was no change in glycolysis relative to control embryos (0.09 pmol/cell/hr, P = 0.3). Expression of ATRX was significantly increased for –P+AOA embryos relative to the control (1.63 v. 1.0, P < 0.007) but did not differ for –P embryos (1.1). This study demonstrates that direct perturbations of mitochondrial function in the embryo compromises its metabolic regulation and blastocyst development, and the expression of the epigenetic modulator ATRX. Further studies are underway to elucidate the implications of disrupted metabolic control and this epigenetic modulator on pregnancy outcomes.
A multi-sensor system for measuring bovine embryo metabolism
