1 INSULIN TREATMENT DURING IN VITRO OOCYTE MATURATION LEADS TO DIFFERENT GENE EXPRESSION AND METHYLATION PATTERNS OF KEY GENES ASSOCIATED WITH METABOLISM AND STEROID SYNTHESIS IN THE BOVINE BLASTOCYST

Abstract

Obesity and overfeeding are common causes for female infertility, leading to insulin resistance and hyperinsulinemia and associated with an increased risk for type 2 diabetes mellitus (Pasquali et al., http://dx.doi.org/10.1093/humupd/dmg024). We investigated here the effect of insulin during in vitro oocyte maturation on methylation changes in bovine Day 8 blastocysts (BC8) and focused on methylation patterns of candidate genes associated with metabolism and steroidogenesis (Day 0 = day of oocyte collection). Abattoir-derived oocytes (n = 882) were in vitro matured for 22 h with 2 different insulin concentrations, INS10 (10 µg mL⁻¹) and INS0.1 (0.1 µg mL⁻¹) or without insulin (INS0, control). Subsequently, IVF and IVC were performed to equal standardized conditions for all groups. Parallel genomic DNA and total RNA extraction (AllPrepDNA/RNA micro kit, cat no. 80284, Qiagen®, Valencia, CA, USA) from pools of 10 frozen (−80°C) BC8 was followed by transcriptome and epigenome analysis (Laskowski et al., http://dx.doi.org/10.1071/RD15315). An empirical Bayes moderated t-test and the ‘limma’ package in R (www.r-project.org) were used to search for differentially expressed genes between the control and the insulin groups. Analysis of the epigenome by using a specific pipeline, described by Shojaei Saadi et al. (2014 BMC Genomics 15, 451), showed that 7632 and 3914 regions were hypomethylated in the INS0.1 and INS10 v. INS0, whereas 6026 and 8504 regions were hypermethylated in INS0.1 and INS10 v. INS0. Combining epigenetic and transcriptomic data, we found that high methylation and low expression or the reverse (low methylation and high expression) were observed for a set of 14 and 11 genes for INS0.1 and INS10 respectively. Most of these genes are associated with lipid metabolism, steroid synthesis, and oxidative stress. Further investigation of the localization of differentially methylated regions (DMR) in genes showed that the conservation odds (methylation) was in general higher in coding regions and CpG islands than in noncoding regions. We observed a large overlap of DMR in the 2 insulin groups compared with controls (3233 common DMR). These numerous changes illustrate the potential unfavourable effects of elevated insulin during maturation leading to alteration of the methylation patterns of the early embryo. This model may help us better understand the mechanisms by which metabolic disorders observed pre-conception can affect embryonic development and subsequent health of the offspring. Our results based on changes in transcriptome or epigenome did show that insulin challenge during maturation leads to postponed effects associated with steroidogenesis, lipid metabolism and oxidative stress in the BC8. By this early stage, if persistent, specific changes in the expression and methylation patterns of genes associated to hyperinsulinemia may decrease the developmental potential of early embryos or could be responsible for subsequent pathologies.

This study was funded by FORMAS.