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

Abstract

Genetically engineered pigs serve as excellent biomedical and agricultural models. To date, the most reliable way to generate genetically engineered pigs is via somatic cell nuclear transfer (SCNT), however, the efficiency of cloning in pigs is low (1–3%). Somatic cells such as fibroblasts frequently used in nuclear transfer utilize the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation for efficient energy production. The metabolism of somatic cells contrasts with cells within the early embryo, which predominately use glycolysis. We hypothesized that fibroblast cells could become blastomere‐like if mitochondrial oxidative phosphorylation was inhibited by hypoxia and that this would result in improved in vitro embryonic development after SCNT. In a previous study, we demonstrated that fibroblasts cultured under hypoxic conditions had changes in gene expression consistent with increased glycolytic/gluconeogenic metabolism. The goal of this pilot study was to determine if subsequent in vitro embryo development is impacted by cloning porcine embryonic fibroblasts cultured in hypoxia. Here we demonstrate that in vitro measures such as early cleavage, blastocyst development, and blastocyst cell number are improved (4.4%, 5.5%, and 17.6 cells, respectively) when donor cells are cultured in hypoxia before nuclear transfer. Survival probability was increased in clones from hypoxic cultured donors compared to controls (8.5 vs. 4.0 ± 0.2). These results suggest that the clones from donor cells cultured in hypoxia are more developmentally competent and this may be due to improved nuclear reprogramming during somatic cell nuclear transfer.

Current applications of SCNT in advanced breeding and genome editing in livestock

“…Primary cell cultures have finite lifespan and after a given number of doublings will reach a stage where they undergo aging and stop replication, however, we are far from a full understanding of what are the ideal requirements and the reasons for success or failure in obtaining offspring after SCNT. Culture conditions, doubling numbers, oxygen tension (Mordhorst et al 2019) etc. can all contribute to the selection during in vitro culture of a particular cell population or a subpopulation that influence the status of the chromatin and most importantly its susceptibility to be reprogrammed after nuclear transfer.…”

Manipulating the Epigenome in Nuclear Transfer Cloning: Where, When and How

“…Hypoxia inducible factor 1 subunit alpha (HIF1A), a transcription factor that allows for cell survival at low oxygen tension, promotes a metabolic switch from somatic cell specific oxidative phosphorylation to glycolysis used by early embryos [ 119 , 120 ]. Stabilization of HIF1A by treatment of donor cells with cobalt chloride (CoCl 2 ) upregulated mRNA abundances of glycolytic enzymes and improved development of porcine cloned embryos to the blastocyst stage [ 121 ].…”

Applications of omics and nanotechnology to improve pig embryo production in vitro

“…Furthermore, donor cells exposed to hypoxic culture conditions (1.25% O 2 ) demonstrated increased abundance of transcripts involved in glycolysis and smaller mitochondria compared to control cells cultured at 5% O 2 , indicating that this method may be effective for inducing the WE (Mordhorst, Murphy, Schauflinger, et al, ). When hypoxia‐treated donor cells were used for SCNT, reconstructed embryos had increased development to the blastocyst stage, total cell numbers in the blastocysts, and survival probability after transfer compared to control donor cells (Mordhorst et al, ).…”