Risks of using mitoquinone during in vitro maturation and its potential protective effects against lipotoxicity-induced oocyte mitochondrial stress.

PURPOSE: Oxidative stress and mitochondrial dysfunction play central roles in reduced oocyte quality and infertility in obese patients. Mitochondria-targeted treatments containing co-enzyme Q10 such as mitoquinone (MitoQ) can increase mitochondrial antioxidative capacity; however, their safety and efficiency when supplemented to oocytes under lipotoxic conditions have not been described. METHODS: We tested the effect of different concentrations of MitoQ or its cationic carrier (TPP) (0, 0.1, 0.5, 1.0 µM each) during bovine oocyte IVM. Then, we tested the protective capacity of MitoQ (0.1 µM) against palmitic acid (PA)-induced lipotoxicity and mitochondrial dysfunction in oocytes. RESULTS: Exposure to MitoQ, or TPP only, at 1 µM significantly (P<0.05) reduced oocyte mitochondrial inner membrane potential (JC-1 staining) and resulted in reduced cleavage and blastocyst rates compared with solvent control. Lower concentrations of MitoQ or TPP had no effects on embryo development under control (PA-free) conditions. As expected, PA increased the levels of MMP and ROS in oocytes (CellROX staining) and reduced cleavage and blastocyst rates compared with the controls (P<0.05). These negative effects were ameliorated by 0.1 µM MitoQ. In contrast, 0.1 µM TPP alone had no protective effects. MitoQ also normalized the expression of HSP10 and TFAM, and partially normalized HSP60 in the produced blastocysts, indicating at least a partial alleviation of PA-induced mitochondrial stress. CONCLUSION: Oocyte exposure to MitoQ may disturb mitochondrial bioenergetic functions and developmental capacity due to a TPP-induced cationic overload. A fine-tuned concentration of MitoQ can protect against lipotoxicity-induced mitochondrial stress during IVM and restore developmental competence and embryo quality.

Rescue Potential of Supportive Embryo Culture Conditions on Bovine Embryos Derived from Metabolically Compromised Oocytes.

Elevated non-esterified fatty acid (NEFA), predominantly palmitic acid (PA), concentrations in blood and follicular fluid are a common feature in maternal metabolic disorders such as obesity. This has a direct negative impact on oocyte developmental competence and the resulting blastocyst quality. We use NEFA-exposure during bovine oocyte in vitro maturation (IVM) as a model to mimic oocyte maturation under maternal metabolic stress conditions. However, the impact of supportive embryo culture conditions on these metabolically compromised zygotes are not known yet. We investigated if the addition of anti-apoptotic, antioxidative and mitogenic factors (namely, Insulin-Transferrin-Selenium (ITS) or serum) to embryo culture media would rescue development and important embryo quality parameters (cell proliferation, apoptosis, cellular metabolism and gene expression patterns) of bovine embryos derived from high PA- or high NEFA-exposed oocytes when compared to controls (exposed to basal NEFA concentrations). ITS supplementation during in vitro culture of PA-exposed oocytes supported the development of lower quality embryos during earlier development. However, surviving blastocysts were of inferior quality. In contrast, addition of serum to the culture medium did not improve developmental competence of PA-exposed oocytes. Furthermore, surviving embryos displayed higher apoptotic cell indices and an aberrant cellular metabolism. We conclude that some supportive embryo culture supplements like ITS and serum may increase IVF success rates of metabolically compromised oocytes but this may increase the risk of reduced embryo quality and may thus have other long-term consequences.

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

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.

Polyunsaturated fatty acids influence offspring sex ratio in cows.

Dietary polyunsaturated fatty acids (PUFAs) can influence fertility in farm animals. Some evidence in mice and sheep have suggested that PUFAs may influence offspring sex ratio, which may have significant value for cattle production. To test this hypothesis, three groups of Holstein cows were supplemented with either 0%, 3% or 5% protected fat (PF) in the form of calcium salt of fatty acids (rich in omega-6) from 14-21 days pre-partum until conception. Proven-fertile frozen semen from the same ejaculate was used for insemination. Calf sex recorded at birth was 8/19 (42.1%) male offspring in the control group, increasing to 14/20 (70%, P > 0.05) and 17/20 (85%, P < 0.05) in 3% and 5% PF, respectively. To test if this effect was caused by a direct influence on the oocyte, we supplemented bovine cumulus oocyte complexes during in vitro maturation with either omega-3 alpha-linolenic acid (ALA), omega-6 linoleic acid (LA) or trans-10, cis-12 conjugated linoleic acid (CLA). Sex ratio of the produced transferable embryos was determined using PCR of SRY gene. Similar to the in vivo results, sex ratio was skewed to the male side in the embryos derived from LA- and CLA-treated oocytes (79% and 71%) compared to control and ALA-treated oocytes (44% and 54%, respectively). These results indicate that both dietary and in vitro supplementation of omega-6 PUFAs can skew the sex ratio towards the male side in cattle. Further experiments are required to confirm this effect on a larger scale and to study the mechanisms of action that might be involved.

Alpha-linolenic acid protects the developmental capacity of bovine cumulus-oocyte complexes matured under lipotoxic conditions in vitro.

Elevated concentrations of free fatty acids (FFAs), predominantly palmitic, stearic, and oleic acids (PSO), exert detrimental effects on oocyte developmental competence. This study examined the effects of omega-3 alpha-linolenic acid (ALA) during in vitro oocyte maturation (IVM) in the presence of PSO on subsequent embryo development and quality, and the cellular mechanisms that might be involved. Bovine cumulus-oocyte complexes (COCs) were supplemented during IVM with ALA (50 µM), PSO (425 µM), or PSO+ALA. Compared with FFA-free controls (P < 0.05), PSO increased embryo fragmentation and decreased good quality embryos on day 2 postfertilization. Day 7 blastocyst rate was also reduced. Day 8 blastocysts had lower cell counts and higher apoptosis but normal metabolic profile. In the PSO group, cumulus cell (CC) expansion was inhibited with an increased CC apoptosis while COC metabolism was not affected. Mitochondrial inner membrane potential (MMP; JC-1 staining) was reduced in the CCs and oocytes. Heat shock protein 70 (HSP70) but not glucose-regulated protein 78 kDa (GRP78, known as BiP; an endoplasmic reticulum stress marker) was upregulated in the CCs. Higher reactive oxygen species levels (DCHFDA staining) were detected in the oocytes. In contrast, adding ALA in the presence of PSO normalized embryo fragmentation, cleavage, blastocyst rates, and blastocyst quality compared to controls (P > 0.05). Combined treatment with ALA also reduced CC apoptosis, partially recovered CC expansion, abrogated the reduction in MMP in the CCs but not in the oocytes, and reduced BiP and HSP70 expression in CCs, compared with PSO only (P < 0.05). In conclusion, ALA supplementation protected oocyte developmental capacity under lipotoxic conditions mainly by protecting cumulus cell viability.

Protective effects of antioxidants on linoleic acid-treated bovine oocytes during maturation and subsequent embryo development.

Linoleic acid (LA; n-6, 18:2) is the most abundant polyunsaturated fatty acid in the ovarian follicular fluid and is known to inhibit oocyte maturation and its subsequent development. In the present study, we investigated how its effects on cumulus cell expansion, oocyte nuclear maturation, and blastocyst development are altered by supplementation of the media with vitamin E (VE; 100 µM) and glutathione peroxidase (GPx; 1 µM) either alone or in combination, and whether it has any effect on the mRNA expression of GPx1, GPx4, or superoxide dismutase (SOD2) in the bovine cumulus oocyte complexes (COCs). LA supplementation of the culture media significantly (P ≤ 0.05) reduced the percentage of COCs exhibiting full cumulus cell expansion and the percentage of oocytes reaching metaphase II stage, and lowered the blastocyst rate compared with controls. And these inhibitory effects were associated with a reduction in the relative mRNA expression of GPx1 and SOD2 but not of GPx4 compared with controls. However, VE and GPx, both alone and in combination, completely abrogated the inhibitory effects of LA on nuclear maturation of oocytes and blastocyst rate but failed to do so for cumulus cell expansion. In conclusion, these data suggest that the detrimental effects of LA on oocyte developmental competence are mediated, at least in part, by a reduction in GPx1 and SOD2 mRNA expression. Moreover, VE and GPx may provide protection to most of the inhibitory effects of LA.