30 OXYGEN DEPRIVATION DOES NOT FURTHER AUGMENT MITOCHONDRIAL MEMBRANE POTENTIAL IN PHARMACOLOGICALLY TREATED FIBROBLASTS FOR USE IN SOMATIC CELL NUCLEAR TRANSFER

Abstract

Somatic cells commonly used in nuclear transfer primarily utilise the tricarboxylic acid cycle and cellular respiration for energy production. Comparatively, the metabolism of somatic cells contrasts that of cells within early embryos, which predominantly use glycolysis and exhibit Warburg Effect (WE)-like characteristics. We hypothesised that fibroblast cells can become more blastomere-like if driven either pharmacologically or by oxygen constraint and could result in improved in vitro embryonic development after somatic cell nuclear transfer (SCNT). The pharmaceuticals used (PS48 and CPI-613) should decrease mitochondrial use of the tricarboxylic acid (TCA) cycle and promote the PI3K pathway, respectively. Furthermore, we hypothesised that oxygen constraint (1.3%) would hinder TCA cycle activity and promote glycolysis. The goal was to achieve a WE-like effect in donor cells before nuclear transfer (NT) by treating Day 35 porcine fetal fibroblasts with CPI-613 (100 µM), PS48 (10 µM), both drugs combined (MIX), or as controls (CON, 0 µM) for 7 days under stepwise oxygen constraint (OC; 1.3%) or under normal conditions (ON; 5%). Three biological replicates were collected and data were analysed for main effect of treatment via GLM procedure of SAS 9.4 (SAS Institute Inc., Cary, NC, USA). To determine if our treatments affected mitochondria respiratory capacity (thereby TCA cycle capability) within embryos, we measured mitochondrial membrane potential (Δψ_m) using JC-10, a biphasic cationic dye. Mitotracker green (MTG) was used to estimate mitochondrial quantity. The percentage of cells with low Δψ_m was increased (P = 0.02) with any CPI or MIX treatment (treatments ≥ 95%) compared with OC-PS48 and both control (ON and OC) treatments (treatments ≥ 77.4%), whereas ON-PS48 had an intermediate level (90.4%; error = 4.9%). Contrary to our prediction, MTG intensity was lower across all ON treatments compared with OC treatments (NO treatments ≤ 736 AU v. OC treatments ≥ 872 AU; error = 23 AU; P < 0.01). Regardless of oxygen level, controls and PS48 treatments yielded the highest percentages of viable cells (treatments ≥ 94%) and OC-CPI and NO-MIX the lowest (treatments ≤ 86%) with NO-CPI and OC-MIX being intermediate (treatments ≥ 90%; error = 3%; P < 0.01). Oxygen constraint did not promote a reduction in mitochondrial membrane potential in pharmacologically treated fibroblasts. Additionally, intensity of MTG was increased in fibroblasts cultured under oxygen constraint compared with those cultured in 5% oxygen. Our results warrant further investigation of the mitochondrial changes occurring with oxygen deprivation in donor-cells. Experiments are underway to determine if gene expression in cells treated pharmacologically and with oxygen constraint are augmented, and whether these treatments will result in better development after SCNT.

This study was funded by Food for the 21 st Century and NIH R01HD080636.