Glutaminolysis provides TCA cycle intermediates necessary for proliferation in the conceptus trophectoderm of pigs
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During the peri-implantation period, sheep and pig conceptuses (embryo and placental membranes) rapidly elongate from spherical to tubular to filamentous forms. In concert with trophectoderm outgrowth during ovine conceptus elongation, binucleate cells (BCs) begin to differentiate from the mononuclear trophoblast cells and migrate to the endometrial luminal epithelium (LE) to form syncytial plaques. These proliferating and migrating cells rapidly consume available O2 and nutrients, resulting in metabolic stress for implanting conceptuses. Metabolism usually occurs through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. However, cells within metabolically restrictive environments, such as immune and tumor cells, switch from oxidative phosphorylation to glycolysis to provide biosynthetic intermediates that feed into other metabolic pathways that are important for cell proliferation and survival. One metabolic change that occurs in proliferating immune and tumor cells is an increase in serine biosynthesis followed by one-carbon metabolism in which glycolysis-derived 3-phosphoglycerate (3-PG) is converted to serine and then formate, a precursor for purines and thymidine that are essential for nucleotide synthesis and cell proliferation. We hypothesized that serine biosynthesis and one-carbon metabolism support proliferation and migration of conceptus trophectoderm within a hypoxic, metabolically restricted uterine environment, and examined sites of conceptus implantation of sheep and pigs by H&E and immunofluorescence staining to determine: 1) if conceptuses are exposed and respond to a hypoxic environment; 2) if mechanistic target of rapamycin (mTOR) is activated in the conceptus trophectoderm; 3) if enzymes that convert glucose to serine, including phosphoglyceride dehydrogenase (PHGDH), phophoserine aminotransferase (PSAT), and phophoserine phosphatase (PSPH) are expressed in the uterine endometrium and conceptus; and 4) if enzymes of one-carbon metabolism that convert serine to formate, including serine hydroxymethyltransferase 2 (SHMT2) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), are expressed in the conceptus trophectoderm. Our results provide insights into how the implanting conceptuses of sheep and pigs adapt metabolically to a hypoxic environment because we observed that: 1) red blood cells accumulate within the developing vasculature of the endometrial stroma, indicating a high demand for oxygen at implantation sites of both sheep and pigs; 2) hypoxia inducible factor 1 alpha (HIF1α) is expressed by the endometrial LE and conceptus trophectoderm of both sheep and pigs; 3) HIF1α and phospho-ribosomal protein s6 (p-RPS6) expression co-localize within the conceptus trophectoderm suggesting a metabolic link between hypoxia and activation of mTOR in the conceptus trophectoderm of both sheep and pigs; 4) PHGDH and PSPH are expressed by the endometrial LE of both sheep and pigs; and 5) SHMT2 and MTHFD2 are expressed by the proliferating trophectoderm of pig conceptuses and expressed by the migrating BCs of sheep conceptuses. We conclude that glucose can be converted to serine within the endometrial LE, serine can be transported into conceptus trophectoderm, and mTOR-HIF1α signaling can induce expression of SHMT2 and MTHFD2 in conceptus trophectoderm to support trophectoderm cell proliferation and migration within the a hypoxic, metabolically restricted environment.
