Mitochondria-targeted therapy rescues development and quality of embryos derived from oocytes matured under oxidative stress conditions: A bovine in vitro model

Abstract

STUDY QUESTION: Can we use a mitochondrial-targeted antioxidant (Mitoquinone) during in vitro embryo culture to rescue developmental competence of oocytes matured under lipotoxic conditions, exhibiting mitochondrial dysfunction and oxidative stress? SUMMARY ANSWER: Supplementation of embryo culture media with Mitoquinone reduced oxidative stress and prevented mitochondrial uncoupling in embryos derived from metabolically compromised oocytes in vitro, leading to higher blastocyst rates and lower blastomeric apoptosis. WHAT IS KNOWN ALREADY: Maternal metabolic disorders, such as obesity and type-II diabetes are associated with hyperlipidemia and elevated free fatty acid (FFA) concentrations in the ovarian follicular fluid (FF). Oocyte maturation under these lipotoxic conditions results in increased oxidative stress levels, mitochondrial dysfunction, reduced developmental competence and disappointing IVF results. STUDY DESIGN, SIZE, DURATION: A well-described bovine oocyte IVM model was used, where a pathophysiologically relevant elevated FF concentrations of palmitic acid (PA; 150 μM or 300 μM) were added to induce oxidative stress. After fertilization (Day 0, D0), zygotes were in vitro cultured (IVC, from D1 to D8) in standard fatty acid-free media in the presence or absence of Mitoquinone or its carrier triphenyl-phosphonium. PARTICIPANTS/MATERIALS, SETTING, METHODS: Embryo cleavage and fragmentation (D2) and blastocyst rates (D8) were recorded. Mitochondrial activity and oxidative stress in cleaved embryos at D2 were determined using specific fluorogenic probes and confocal microscopy. D8 blastocysts were used to (i) examine the expression of marker genes related to mitochondrial unfolded protein responses (UPRmt; HSPD1 and HSPE1), mitochondrial biogenesis (TFAM), endoplasmic reticulum (ER) UPR (ATF4, ATF6 and BiP) and oxidative stress (CAT, GPX1 and SOD2) using real time RT-PCR; (ii) determine cell differentiation and apoptosis using CDX-2 and cleaved caspase-3 immunostaining; and (iii) measure mtDNA copy numbers. This was tested in a series of experiments with at least three independent replicates for each, using a total of 2525 oocytes. Differences were considered significant if a P value was <0.05 after Bonferroni correction. MAIN RESULTS AND THE ROLE OF CHANCE: Exposure to PA during IVM followed by culture under control conditions resulted in a significant increase in oxidative stress in embryos at D2. This was associated with a significant reduction in mitochondrial inner membrane potential (uncoupling) compared with solvent control (P < 0.05). The magnitude of these effects was PA-concentration dependent. Consequently, development to the blastocysts stage was significantly hampered. Surviving blastocysts exhibited high apoptotic cell indices and upregulated mRNA expression indicating persistent oxidative stress, mitochondrial and ER UPRs. In contrast, supplementation of PA-derived zygotes with Mitoquinone during IVC (i) prevented mitochondrial uncoupling and alleviated oxidative stress at D2; and (ii) rescued blastocyst quality; normalized oxidative stress and UPR related genes and apoptotic cell indices (P > 0.01 compared with solvent control). Mitoquinone also improved blastocyst rate in PA-exposed groups, an effect that was dependent on PA concentration. LARGE SCALE DATA: N/A LIMITATIONS, REASONS FOR CAUTION: This is a fundamental study performed using a bovine in vitro model using PA-induced lipotoxicity during oocyte maturation. PA is the most predominant FFA in the FF that is known to induce lipotoxicity; however, in vivo maturation in patients suffering from maternal metabolic disorders involve more factors that cannot be represented in one model. Nevertheless, focusing on the carryover oxidative stress as a known key factor affecting developmental competence, and considering the novel beneficial rescuing effects of Mitoquinone shown here, we believe this model is of high biological relevance. WIDER IMPLICATIONS OF THE FINDINGS: Human oocytes collected for IVF treatments from patients with maternal metabolic disorders are vulnerable to lipotoxicity and oxidative stress during in vivo maturation. The results shown here suggest that mitochondrial targeted therapy, such as using Mitoquinone, during IVC may rescue the developmental competence and quality of these compromised oocytes. After further clinical trials, this may be a valuable approach to increase IVF success rates for infertile patients experiencing metabolic disorders. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by a BOF/KP grant number 34399, from the University of Antwerp, Belgium. W.F.A.M. was supported by a postdoctoral fellowship from the Research Foundation-Flanders (FWO), grant number 12I1417N, Antwerp, Belgium. The Leica SP 8 confocal microscope used in this study was funded by the Hercules Foundation of the Flemish Government (Hercules grant AUHA.15.12). All authors have no financial or non-financial competing interests to declare.