3 OXIDATIVE STRESS INDUCED CHANGES IN EPIGENETIC MODIFYING GENE mRNA IN PRE-IMPLANTATION IN VITRO BOVINE EMBRYOS
Authors: Burroughs C. A. , Williamson G. L. , Golding M. C. , Long C. R. 
Published: December 2012

In vitro embryo culture systems introduce a myriad of environmental changes to embryos when compared with in vivo development. Accordingly, in vitro culture has been associated with decreased embryo development rates and alterations to the fetal epigenome. We hypothesized that an interaction exists between oxidative stress and the expression of key epigenetic modifying enzymes. To test this hypothesis, we used quantitative PCR to measure transcript levels of several genes known to be associated with oxidative stress and epigenetic modifications during bovine in vitro pre-implantation development. Epigenetic modifiers examined included histone deacetylases (SIRT1, SIRT6), whose actions result in gene repression; a histone demethylase (JMJD1A) associated with gene activation; and DNA hydroxylases (TET1-3), which have been associated with conversion of 5-methylcytosine to 5-hydroxy-methylcytosine. Bovine ova were matured, fertilized, and cultured in vitro according to standard laboratory procedures. At 18 h post-fertilization, cumulus cells were removed and presumptive zygotes were cultured in 5% CO2 and air (high O2), or 5% CO2, 5% O2 and 90% N (low O2). Groups of 16-cell- (n = 20) and blastocyst- (n = 15) stage embryos were collected, RNA was extracted using an RNeasy Mini Kit, and cDNA was generated using qScript reagents. Quantitative PCR was performed using Perfecta SYBR to determine transcript levels of superoxide dismutase 2, mitochondrial (SOD2), lactate dehydrogenase A (LDHA), hypoxia-inducible factor 1 alpha (HIF1A), hypoxia-inducible factor 2 alpha (HIF2A), histone deacetylase 1 (HDAC1), jumonji domain containing 1A (JMJD1A), sirtuin 1 (SIRT1), sirtuin 6 (SIRT6), tet oncogene 1 (TET1), tet oncogene 2 (TET2), and tet oncogene 3 (TET3) in 3 replicates (each measured in triplicate). Data were analyzed using the comparative Ct method normalized to the geometric mean of 2 endogenous control genes [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), succinate dehydrogenase (SDHA)]. Statistical analyses were performed using a paired t-test comparing the relative expression of each gene in high O2 with low O2 within each individual embryo stage. Blastocyst development rates were 35% (n = 710 oocytes) for low O2 and 19% for high O2 (n = 1221 oocytes; P < 0.001). The HIF2A transcript levels were elevated at the 16-cell stage in high-O2 embryos (4.26 ± 0.55 v. 1.22 ± 0.22; P < 0.01), but were not different at the blastocyst stage. Elevated HIF2A was likely in response to oxidative stress and was associated with elevated levels of JMJD1A (1.73 ± 0.18 v. 1.15 ± 0.19), SIRT1 (1.68 ± 0.20 v. 1.09 ± 0.16), SIRT6 (2.30 ± 0.34 v. 1.07 ± 0.13), and TET2 (3.44 ± 0.89 v. 1.23 ± 0.19; P < 0.05) in high-O2 16-cell embryos. The JMJD1A (1.31 ± 0.17 v. 1.05 ± 0.10), SIRT1 (1.45 ± 0.24 v. 1.05 ± 0.11), and TET1 (1.45 ± 0.19 v. 1.02 ± 0.07) transcripts were also elevated in high-O2 blastocysts, whereas TET3 transcript levels were lower (0.69 ± 0.12) than in low O2 (1.07 ± 0.14; P < 0.05). Genes associated with epigenome modification and with oxidative stress were altered in response to the high oxygen tension. The lower blastocyst rate observed in the high-O2 group may be due to the damaging effects of oxidative stress and this stress alters transcript levels associated with the epigenome. These data suggest that embryos surviving oxidative stress may exhibit epigenetic changes that could influence subsequent development.