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

Abstract

The Warburg effect is characterized by decreased mitochondrial oxidative phosphorylation and increased glycolytic flux in adequate oxygen. The preimplantation embryo has been described to have characteristics of the Warburg effect, including similar changes in gene expression and mitochondria, which are more rudimentary in appearance. We hypothesized hypoxia would facilitate anaerobic glycolysis in fibroblasts thereby promoting gene expression and media metabolite production reflecting the Warburg effect hallmarks in early embryos. Additionally, we speculated that hypoxia would induce a rudimentary small mitochondrial phenotype observed in several cell types evidenced to demonstrate the Warburg effect. While many have examined the role hypoxia plays in pathological conditions, few studies have investigated changes in primary cells which could be used in somatic cell nuclear transfer. We found that cells grown in 1.25% O2 had normal cell viability and more, but smaller mitochondria. Several hypoxia-inducible genes were identified, including seven genes for glycolytic enzymes. In conditioned media from hypoxic cells, the quantities of gluconolactone, cytosine, and uric acid were decreased indicating higher consumption than control cells. These results indicate that fibroblasts alter gene expression and mitochondria to compensate for hypoxic stress and maintain viability. Furthermore, the metabolic changes observed, making them more similar to preimplantation embryos, could be facilitating nuclear reprogramming making these cells more amendable to future use in somatic cell nuclear transfer.

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

“…Overall these changes lead to a 2.5‐fold increase in system efficiency (blastocyst development rate * fetal survival rate). In this study, our Day 35 pregnancy rate was 50% (6 of 12 surrogate gilts became pregnant) which is higher than previous experiments in our laboratory involving the same personnel (41.6% and 28.6%; Mordhorst, Murphy, Schauflinger, et al, ) through the average number of fetuses per litter was higher compared to the current study (5.9 vs. 4.8 fetuses/litter). One limitation of this study is that at the present time, no piglets have been delivered to term using hypoxic donors.…”

“…Porcine fetal fibroblast cell lines collected from dorsal regions (fetal shoulders to the rump/tail head) used in the study were established from d 35 pregnancies and utilized in previous experiments in our laboratory (Mordhorst, Murphy, Schauflinger, et al, ; Prather, Rowland et al, ). A cryogenic vial of fibroblasts (0.5 ml aliquots; ~1.5 million/ml in media containing 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide) was defrosted from liquid nitrogen storage for every replicate in the experiment.…”

“…Methods for SCNT were previously reported by (Mordhorst, Murphy, Schauflinger, et al, ). Sow‐derived oocytes were purchased from DeSoto Biosciences (Seymour, TN) and shipped overnight in maturation medium (90% M199 and 10% follicular fluid supplemented with 0.57 mM cysteine, 5 μg/ml insulin, 10 ng/ml epidermal growth factor, 5 μg/ml LH and FSH).…”

“…Blastocyst‐stage embryos ( N = 16–20 per SCNT fibroblast donor culture treatment) were surgically transferred into the oviductal ampullary‐isthmic junction of surrogate gilt recipients (3–5 days postobserved estrus). Surgical embryo transfer technique was conducted as previously established within our laboratory (Lai & Prather, ; Mordhorst, Murphy, Schauflinger, et al, ). For embryo transfer experiments, two surrogate gilts were used in each replicate (six replicates; 12 gilts) in a complete‐block type fashion where between the gilts each cell line (two of the three different lines utilized) and donor cell culture treatment (hypoxia and control) were used.…”

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

“…Treatment with the compounds did not promote proliferation of the donor cells but increased secretion of pyruvate into the medium which is a characteristic of the reverse WE (Mordhorst, Murphy, Ross, et al, ; Pavlides et al, ). 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, ).…”