Pharmacologic treatment of donor cells induced to have a Warburg effect‐like metabolism does not alter embryonic development in vitro or survival during early gestation when used in somatic cell nuclear transfer in pigs

Abstract

Somatic cell nuclear transfer is a valuable technique for the generation of genetically engineered animals, however, the efficiency of cloning in mammalian species is low (1-3%). Differentiated somatic cells commonly used in nuclear transfer utilize the tricarboxylic acid cycle and cellular respiration for energy production. Comparatively the metabolism of somatic cells contrasts that of the cells within the early embryos which predominately use glycolysis. Early embryos (prior to implantation) are evidenced to exhibit characteristics of a Warburg Effect (WE)-like metabolism. We hypothesized that pharmacologically driven fibroblast cells can become more blastomere-like and result in improved in vitro embryonic development after SCNT. The goals were to determine if subsequent in vitro embryo development is impacted by (1) cloning pharmacologically treated donor cells pushed to have a WE-like metabolism or (2) culturing non-treated donor clones with pharmaceuticals used to push a WE-like metabolism. Additionally, we investigated early gestational survival of the donor-treated clone embryos. Here we demonstrate that in vitro development of clones is not hindered by pharmacologically treating either the donor cells or the embryos themselves with CPI, PS48, or the combination of these drugs. Furthermore, these experiments demonstrate that early embryos (or at least in vitro produced embryos) have a low proportion of mitochondria which have high membrane potential and treatment with these pharmaceuticals does not further alter the mitochondrial function in early embryos. Lastly, we show that survival in early gestation was not different between clones from pharmacologically induced WE-like donor cells and controls.

Improvement of in vitro and early in utero porcine clone development after somatic donor cells are cultured under hypoxia

“…It was desirable that this ‘reprogramming’ induction be through a simple mechanism that could be applied to any cell line, such as altering the metabolism during culture via hypoxia. Previously our laboratory reported that oxygen restriction in porcine fetal fibroblasts (the same line used in in vitro experiments in the current study) induced expression of hexokinases 1 and 2 , glucose‐6‐phosphate isomerase , glyceraldehyde‐3‐phosphate dehydrogenase , triose phosphate isomerase 1, aldolase, fructose‐bisphosphate C , and phosphoglycerate kinase ; all enzymes involved in glycolysis (Mordhorst, Murphy, Ross, et al, ). In addition, expression of pyruvate dehydrogenase kinase 1 was also induced, this kinase is evidenced to be a key player in thwarting the entrance of pyruvate into the TCA cycle thereby effectively decreasing mitochondrial oxidative phosphorylation (Papandreou, Cairns, Fontana, Lim, & Denko, ).…”

“…In addition, expression of pyruvate dehydrogenase kinase 1 was also induced, this kinase is evidenced to be a key player in thwarting the entrance of pyruvate into the TCA cycle thereby effectively decreasing mitochondrial oxidative phosphorylation (Papandreou, Cairns, Fontana, Lim, & Denko, ). Hypoxia treatment results in metabolism being shifted to become more blastomere‐like (Mordhorst, Murphy, Ross, et al, ; Redel et al, ).…”

“…Further exploration is necessary to pinpoint effective duration of hypoxic culture and whether stepwise decreases in oxygen concentration are necessary. In this study, we used a hypoxic culture method which we knew to be effective in inducing a hypoxic cellular phenotype and changes in gene expression (Mordhorst, Murphy, Ross, et al, ).…”

“…In both experiments gestational Day 35 fetal fibroblasts were used, however different cell lines were used. In Experiment 1 a single cell line was used in SCNT (BRM3P1; Mordhorst, Murphy, Ross, et al, ). In embryo transfer experiments, cell lines proven to produce live offspring were used to distinguish (via genotyping; Figure ) which donor cell treatments survived in utero.…”

Porcine Fetal-Derived Fibroblasts Alter Gene Expression and Mitochondria to Compensate for Hypoxic Stress During Culture

“…The current study is novel in its approach to program metabolism of donor fibroblasts to a Warburg effect-like state similar to that of preimplantation embryos in an effort to improve somatic cell nuclear transfer. To date, we do not know of any other groups which have tried similar strategies outside of our laboratory (Mordhorst et al, 2018a , 2018b ).…”