150 Changes in pyruvate metabolism alters the epigenetic and molecular maturation of bovine oocytes
Authors: Silva J. V. A. , Ispada J. , Junior A. M. Fonseca , dos Santos E. C. , de Lima C. B. , da Rocha H. C. , Milazzotto M. P. 
Published: January 2021

During invitro maturation (IVM), bovine oocytes undergo important metabolic, epigenetic, and transcriptional changes for the acquisition of developmental competence. Particularly, metabolic changes that alter the availability of cytoplasmic acetyl-CoA, the main substrate for histones acetylation, may alter the epigenetic profile of the oocyte, with consequences for correct molecular maturation. To test this hypothesis, cumulus–oocyte complexes (COCs) were IVM in three experimental groups: Control [IVM medium (TCM-199-Bicarbonate, 10% fetal bovine serum, 1µg mL−1 oestradiol, 10µg mL−1 FSH, and 10µg mL−1 human chorionic gonadotrophin)], DCA (IVM medium supplemented with 1.5mM sodium dichloroacetate, a pyruvate to acetyl-CoA conversion stimulator) and IA (IVM medium supplemented with 5µM sodium iodoacetate, a glycolysis inhibitor). Cumulus cells (CC) and oocytes (Oo) were analysed separately at 24h (mitochondrial activity, MA; MitoTracker Red CMXRos, ThermoFisher Scientific] and at 0, 4, 8, 16, and 24h of IVM [lysine 9 histone 3 acetylation (H3K9ac immunofluorescence) and new transcript synthesis (only CC; Click-iT® RNA, ThermoFisher Scientific). The images were acquired using a fluorescence microscope and analysed by Image J software. The results from at least 3 replicates were compared by Student’s t-test (treatment vs. control) or by ANOVA followed by Tukey’s test (comparison within the same group in different time points) considering P&lt;0.05. As expected, DCA treatment led to an increase in MA in CC and oocytes (CC control vs. DCA, P=0.003; Oo control vs. DCA, P=0.003). In CC, during the first 4h, H3K9ac increased significantly in the treated group and decreased in the control group. At 8, 16, and 24h, both groups presented similar tendencies, although H3K9ac levels remained higher in DCA compared with control at all time points (P&lt;0.001). The synthesis of new transcripts in CC was stimulated by DCA compared with control at 8h (P=0.02) and particularly at 16h (P=0.002), when acetylation levels were at the lowest point. Interestingly, in oocytes, the initial trend was reversed. An increase was observed in the H3K9ac levels of the control group (P=0.014), whereas no difference was observed for DCA in the first 4h. Moreover, although acetylation levels followed a downward tendency with time in both groups, oocytes treated with DCA showed lower H3K9ac levels at 4 and 8h and a higher level at 24h (P=0.04) compared with control. Regarding IA, lower MA were verified in CC whereas oocytes had the opposite profile (CC control vs. IA: P=0.0035; Oc control vs. IA: P&lt;0.001). In CC, this decrease in MA was not accompanied by a decrease in H3K9ac. In contrast, H3K9ac increased compared with the control group at 8 and 16h (control 8h vs. IA 8 h: P=0.019 and control 16h vs. IA 16 h: P=0.019). These changes were accompanied by an increase in the synthesis of new transcripts in the IA group over the time of IVM. Based on these data, we can conclude that changes in pyruvate metabolism caused by manipulation of the IVM system lead to epigenetic and molecular changes in both CC and oocytes.