2 Inhibition of mitochondrial ATP production during in vitro maturation of bovine oocytes alters DNA methylation patterns in mature oocytes and resulting embryos

Maternal metabolic disorders result in mitochondrial dysfunction and epigenetic alterations in developing oocytes and embryos. Also, ATP is important for the biosynthesis of several coenzymes involved in epigenetic regulation. Furthermore, oocyte maturation is a sensitive window for environmental changes, as important epigenetic modifications take place. It is unknown whether the effects of mitochondrial dysfunction during oocyte maturation on epigenetic regulation are ATP dependent. Therefore, we aimed to specifically reduce mitochondrial ATP production in maturing oocytes to assess epigenetic alterations in these oocytes and produced embryos. A bovine in vitro-production model was developed and used, wherein cumulus-oocyte complexes (COCs) were exposed to control media (CONT; TCM-199 with 0.4 mM L-glutamine, 0.2 mM sodium pyruvate, 50 µg/mL gentamicin, 0.1 µM cysteamine and 20 ng/mL epidermal growth factor) or CONT + 5 nM oligomycin A (OM) during in vitro maturation (IVM) (24 h). OM inhibits mitochondrial ATP production. Oocytes were then in vitro fertilised in Fert-TALP + 0.72 U/mL heparin (20 h) and presumptive zygotes were cultured in synthetic oviducal fluid + 2% bovine serum albumin (7 days) (9 replicates, 943 COCs/treatment). Cleavage rates were recorded at 48 h post-insemination (p.i.) (7 replicates) and blastocyst rates at 8 d p.i. (3 replicates). At 24 h IVM, mitochondrial ATP production was assessed in pools of 15 denuded oocytes (5 replicates) using the ATP rate assay kit of the Seahorse XF HS Mini Analyser (Agilent). Also, oocytes (34/treatment, 3 replicates), zygotes (26/treatment, 3 replicates) and morulae (23–30/treatment, 4 replicates) were collected after 24 h IVM, 20 h p.i., and 4.7 d p.i., respectively, and were fixed for 5 mC immunostaining and confocal microscopy to assess global DNA methylation. Developmental competence data were analysed using logistic regression, and numerical data with t-test or Mann-Whitney U test depending on homogeneity of variance. OM exposure during IVM resulted in a small but significant decrease in cleavage rate compared to CONT (75.0 ± 1.9% vs 80.0 ± 1.8%; P = 0.021), while blastocyst rates were not significantly different (37.9 ± 5.8% vs 41.8 ± 2.8%; P = 0.496). Mitochondrial ATP production was significantly reduced in OM-treated oocytes compared to CONT (52.6 ± 6.1 pmol/min vs 80.4 ± 6.1 pmol/min; P = 0.018). Compared with the CONT group, 5 mC grey scale intensity in the OM group was decreased in oocytes (9.8% reduction; P = 0.019) and increased in zygotes (22.3% increase; P < 0.001) and morulae (10.3% increase; P = 0.041). We conclude that DNA methylation during oocyte maturation is dependent on mitochondrial ATP production. In contrast, the increase in DNA methylation during embryo development after OM exposure could be due to compensatory increase in mitochondrial functions. This is still under investigation. For the first time, a direct ATP-dependent link between mitochondrial function and epigenetic regulation has been confirmed in oocytes and early embryos.