Keefer Lab

2014 SSR Annual Conference (Grand Rapids, MI) - Abstract #191:  Carol L. Keefer ; Jennifer L. Colvin ; Brian J. Bequette 

Determination of glycolytic and TCA cycle activity of early cleavage and blastocyst staged bovine embryos employing [13C]-labeled substrates and mass spectrometry                    

Assisted reproductive technologies involve exposure of embryos to artificial nutritional environments which can result in decreased viability. While uptake and metabolism of glucose is known to increase dramatically at the blastocyst stage, how embryos balance energetic and synthetic demands is not clear. Our aim is to determine how embryos balance these processes during preimplantation development. More specifically, we examined how shifting carbohydrate pathway balance (by altering substrate availability) affected metabolic pathway fluxes (13C-based fluxome). Bovine embryos were acquired from ART, Inc. (Madison, WI) at either the cleavage or blastocyst stage. Embryos (n=4-5) were incubated in 20 μl of medium containing tracers under oil at 38.5°C with 6% O2 6% CO2, and 88% N2. Tracer substrates were [13C3] lactate, [13C3]pyruvate, [13C6]glucose, and [13C5]glutamine. Base medium was modified SOF, which was supplemented with either one of the four stable isotope tracer substrates only or a stable isotope tracer substrate plus unlabelled forms of the other three substrates. After 6 h incubation, embryos and spent culture media were stored (-80 C). Metabolites from spent media were extracted, chemically derivatized, and isotopomer enrichments of metabolites determined by gas chromatography-mass spectrometry. Lactate, pyruvate and TCA cycle intermediate equilibrium partners alanine (pyruvate), aspartate (oxaloacetate), and glutamate (α-ketoglutarate) were monitored for calculation of glycolytic and TCA cycle fluxes. When medium contained only [13C6]glucose as substrate, 31% of pyruvate flux was derived from glucose in blastocysts while in cleavage staged embryos only 6% of pyruvate was derived from glucose. When medium contained all four substrates, <1% of pyruvate was derived from glucose for blastocyst and cleavage stage embryos. 13C-Kinetic estimates indicated that addition of all substrates to [13C6]glucose medium dramatically increased the relative flux of the pentose phosphate pathway (PPP) compared to glycolysis for both blastocysts (0.13:1 vs 0.6:1) and early cleavage stage (0.47:1 vs 2.23:1). In contrast, when medium contained only [13C3]lactate as substrate, blastocyst and cleavage stages derived 41-50% of pyruvate flux from lactate, and this contribution fell to 2-9% when all four substrates were present. Surprisingly, cleavage stage embryos did not metabolize any of the four substrates to acetyl-CoA (oxidative metabolism) via the pyruvate dehydrogenase (PDH) reaction, and the metabolism of these four substrates to oxaloacetate via the pyruvate carboxylase (PC) reaction was minor (<0.5%). In comparison, blastocysts derived 10% of acetyl-CoA flux from [13C3] pyruvate when it was the lone substrate and this increased to 37% of acetyl-CoA flux when all four substrates were present. The latter finding is consistent with our 13C-kinetic estimates of the relative fluxes through PDH vs. PC where the addition of all four substrates to the medium increased the relative fluxes from 1.6:1 to 5.2:1. In summary, the results indicate that provision of lactate, pyruvate, glucose, and glutamine to embryos at both stages shifts glucose metabolism toward the PPP and enhances the activity of PDH relative to PC.