The Trophectoderm of Elongating Conceptuses uses Reverse TCA Cycle Flux to Supply Biosynthetic Precursors within a Physiologically Low Oxygen Intrauterine Environment in Pigs.

Abstract Authors: Joe W. Cain^1,2; Guoyao Wu³; Fuller W. Bazer³; Gregory A. Johnson²; Heewon Seo¹

1. Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20742
2. Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX 77843
3. Department of Animal Science, Texas A&M University, College Station, TX 77843

Abstract Text:

Porcine conceptuses develop rapidly through extensive cellular proliferation as they undergo elongation. Tricarboxylic acid (TCA) cycle flux provides biosynthetic precursors for synthesis of lipids, nucleotides, and amino acids. We reported that elongating conceptuses primarily rely on aerobic glycolysis, and most of the glucose-derived carbons are diverted into the production of lactate and into other pathways including the pentose phosphate pathway (PPP), hexosamine biosynthesis, and one-carbon metabolism. This re-routes glucose carbons away from the mitochondrial TCA cycle. Therefore, to maintain TCA cycle flux during aerobic glycolysis, porcine conceptuses utilize glutamine through glutaminolysis as an alternate carbon source. Glutamine is converted into α-ketoglutarate (αKG), which can be subsequently converted to succinate through oxidative reactions. However, under some conditions such as low oxygen levels, cells convert αKG into isocitrate, in the reverse direction of the conventional TCA cycle. Considering the physiologically low oxygen uterine environment (5-8%), it is likely that trophectoderm is in a more reduced state (i.e. a greater ratio of NADH/NAD⁺), reducing the oxidation of acetyl-CoA via the forward TCA cycle and limiting the supply of biosynthetic precursors needed by the elongating conceptus. To meet metabolic needs, glutamine-derived αKG drives the cycle in the reverse direction. Isocitrate dehydrogenase (IDH) catalyzes the interconversion of isocitrate into αKG and exists as three isozymes. IDH1 localizes to both cytosol and peroxisomes, while IDH2 and IDH3, as part of the TCA cycle, are found within the mitochondrial matrix. IDH3 operates in the forward direction of the TCA cycle producing αKG and NADH. NADH is then utilized in the electron transport chain to drive respiration. In contrast, cytosolic IDH1 and mitochondrial IDH2 can catalyze the reaction reversibly in either the forward (oxidative) or the reverse (reductive) direction dependent on environmental conditions. We investigated the expression of these isozymes at the uterine-placental interface during the peri-implantation period in pigs (n=3/day) using immunofluorescence analyses. Mitochondrial IDH3 is weakly detectable in the trophectoderm by Day 15 of pregnancy but expressed apically by Day 20. Mitochondrial IDH2 is only expressed in the uterine luminal epithelium. The lack of expression of IDH2 and IDH3 in the elongating conceptus trophectoderm suggests a disrupted oxidative TCA cycle during conceptus elongation. In contrast, cytosolic IDH1 is detected in the trophectoderm/chorionic epithelium throughout pregnancy, indicating that IDH1 is likely the major isoform used by the trophectoderm of elongating conceptuses. The IDH1-catalyzed reverse reaction depends on external conditions such as high levels of NADPH, which is provided by the PPP that is activated in elongating conceptuses, suggesting that cytosolic NADPH is coupled with reverse TCA cycle flux in the mitochondria during the peri-implantation period. Additionally, ATP citrate synthase (ACLY), an enzyme that irreversibly catalyzes the conversion of citrate into oxaloacetate and acetyl-CoA, is localized in the cytosol of the conceptus trophectoderm. The strong expression of IDH1 and ACLY in the absence of IDH2 and IDH3 in the trophectoderm suggests that porcine conceptuses use reverse TCA cycle reactions to compensate for the lack of oxidative TCA cycle activity and to fulfill biosynthetic demands associated with their rapid elongation. The reverse TCA cycle may provide an adaptive mechanism for sustainment of TCA cycle flux when oxygen is limited during the peri-implantation period in pigs. This work was supported by Agriculture and Food Research Initiative Competitive Grant no. 2020-67015-31136 from the USDA National Institute of Food and Agriculture.