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Application of mitochondrial inhibitors oligomycin, FCCP and antimycin A/rotenone to GV-stage oocytes. (a) Indicates the drug targets of Oligomycin, FCCP, Antimycin A and Rotenone. Oligomycin blocks ATP-synthase, FCCP dissipates the proton gradient between the matrix and inner membrane space, and antimycin A and rotenone inhibit complexes III and I respectively. (b,c) The response of GV-stage CEOs to the sequential addition of 1 μM oligomycin, 5 μM FCC and 2.5 μM A/R, depicted as (b) OCR (pmol/min/oocyte) or (c) as compared to control non-treated oocytes, shown as percentage of the third basal measurement. (mean ± SEM, representative of 12 wells (72 CEOs) treated and 13 wells (78 CEOS) untreated). *Represents p < 0.05 and ****represents p < 0.0001.
Source publication
Mitochondria provide the major source of ATP for mammalian oocyte maturation and early embryo development. Oxygen Consumption Rate (OCR) is an established measure of mitochondrial function. OCR by mammalian oocytes and embryos has generally been restricted to overall uptake and detailed understanding of the components of OCR dedicated to specific m...
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Background
Female fertility declines with age, due to increased chromosomal aneuploidy and possible reduced mitochondrial function in the embryo.
Methods
This review outlines how mitochondrial function in human embryos, as predicted from oxygen consumption rate (OCR) measurements, changes in preimplantation stage, and what factors, particularly ma...
Citations
... The mitochondrial matrix contains essential components such as DNA, ribosomes, enzymes and co-factors needed for the citric acid cycle and oxidative phosphorylation [59,60]. In mature oocytes, while all mitochondria exhibit electron dense matrix and no or very few distinct cristae, these oocytes exhibit a high mitochondrial membrane potential, and detectable O 2 -linked ATP production as shown using Seahorse analysis [61,62]. Thus, the increase in mitochondrial matrix density detected in secondary follicle oocytes may be linked with an increased mitochondrial activity [21]. ...
Diet-induced obesity reduces oocyte quality mainly by impacting oocyte mitochondrial functions. Moreover, maternal obesity is associated with mitochondrial dysfunction in oocytes of their adult offspring. However, these effects were reported only in fully grown oocytes, mainly in the form of abnormal mitochondrial ultrastructure. It is unknown if obesogenic (OB) diets or maternal obesity already impact the primordial and preantral follicles. Considering the long duration and dynamics of folliculogenesis, determining the stage at which oocytes are affected and the extent of the damage is crucial for optimal reproductive management of obese patients and their daughters. Potential interaction between maternal and offspring diet effects are also not described, yet pivotal in our contemporary society. Therefore, here we examined the impact of OB diets on oocyte mitochondrial ultrastructure in primordial and activated preantral follicles in offspring from diet-induced obese or lean mothers. We used an outbred Swiss mouse model to increase the pathophysiological relevance to humans. Female mice were fed control or OB diets for 7 weeks, then mated with control males. Their female offspring were fed control or OB diets after weaning for 7 weeks (2-by-2 factorial design). Adult offspring ovarian sections were examined using transmission electron microscopy. We characterised and classified unique features of oocyte mitochondrial ultrastructure in the preantral follicles. An increase in mitochondrial matrix density was the most predominant change during follicle activation in secondary follicles, a feature that is linked with a higher mitochondrial activity. Maternal obesity increased mitochondrial density already in the primordial follicles suggesting an earlier increase in bioenergetic capacity. Maternal obesity did not induce abberant ultrastructure (abnormalities and defects) in primordial or preantral follicles. In contrast, offspring OB diet increased mitochondrial abnormalities in the primordial follicles. Further investigation of the consequences of these changes on oocyte metabolic regulation and stress levels during folliculogenesis is needed.
... The efficiency of oxidative phosphorylation in terms of ATP formation in mature mitochondria is ~ 40% with the remaining ~ 60% being released as heat by basal and inducible proton leak; for excellent reviews, see Beignon et al. [25] and Bertholet and Kirichok [26], values which, however, can differ in different cells and metabolic states. For example, Muller et al. [27] reported a figure of 20% for the proton leak of in vitro-produced bovine preimplantation embryos measured as oxygen consumption insensitive to oligomycin. Significantly, as well as generating heat, the proton leak reduces the production of ROS and therefore offers a protective mechanism against oxidative stress. ...
A number of factors may impinge on thermal homeostasis in the early embryo. The most obvious is the ambient temperature in which development occurs. Physiologically, the temperature in the lumen of the female tract is typically lower than the core body temperature, yet rises at ovulation in the human, while in an IVF setting, embryos are usually maintained at core body temperature. However, internal cellular developmental processes may modulate thermal control within the embryo itself, especially those occurring in the mitochondria which generate intracellular heat through proton leak and provide the embryo with its own ‘central heating system’. Moreover, mitochondrial movements may serve to buffer high local intracellular temperatures. It is also notable that the preimplantation stages of development would generate proportionally little heat within their mitochondria until the blastocyst stage as mitochondrial metabolism is comparatively low during the cleavage stages. Despite these data, the specific notion of thermal control of preimplantation development has received remarkably scant consideration. This opinion paper illustrates the lack of reliable quantitative data on these markers and identifies a major research agenda which needs to be addressed with urgency in view of laboratory conditions in which embryos are maintained as well as climate change–derived heat stress which has a negative effect on numerous clinical markers of early human embryo development.
... Both spare capacity and proton leak appear to increase with the proportion of males in the population, but do not reach statistical significance ( Fig. 3E and F). Non-mitochondrial respiration is the oxygen consumption associated with other cellular activities and can be due to enzymatic reactive oxygen species (ROS) production, cell-surface oxygen consumption, and non-mitochondrial NADPH oxidases, including cytochrome P450s [51][52][53][54][55]. Interestingly, lower levels of non-mitochondrial respiration were observed in both the him-5(e1490) and him-8(e1489) strains (Fig. 3G). ...
... This reduction was likely driven by a strain difference rather than sex difference because reduced non-mitochondrial respiration was not observed in the N2 male enriched population or fog-2(q71) strain. Non-mitochondrial respiration in cells can be driven by biological processes such as enzymatic ROS production, cell-surface oxygen consumption, and nonmitochondrial NADPH oxidases, including cytochrome P450s [51][52][53][54][55]. him-5(e1490) and him-8(e1489) strains are often used by C. elegans researchers studying males. ...
Background
Sex differences in mitochondrial function have been reported in multiple tissue and cell types. Additionally, sex-variable responses to stressors including environmental pollutants and drugs that cause mitochondrial toxicity have been observed. The mechanisms that establish these differences are thought to include hormonal modulation, epigenetic regulation, double dosing of X-linked genes, and the maternal inheritance of mtDNA. Understanding the drivers of sex differences in mitochondrial function and being able to model them in vitro is important for identifying toxic compounds with sex-variable effects. Additionally, understanding how sex differences in mitochondrial function compare across species may permit insight into the drivers of these differences, which is important for basic biology research. This study explored whether Caenorhabditis elegans , a model organism commonly used to study stress biology and toxicology, exhibits sex differences in mitochondrial function and toxicant susceptibility. To assess sex differences in mitochondrial function, we utilized four male enriched populations (N2 wild-type male enriched, fog-2(q71) , him-5(e1490) , and him-8(e1498) ). We performed whole worm respirometry and determined whole worm ATP levels and mtDNA copy number. To probe whether sex differences manifest only after stress and inform the growing use of C. elegans as a mitochondrial health and toxicologic model, we also assessed susceptibility to a classic mitochondrial toxicant, rotenone.
Results
We detected few to no large differences in mitochondrial function between C. elegans sexes. Though we saw no sex differences in vulnerability to rotenone, we did observe sex differences in the uptake of this lipophilic compound, which may be of interest to those utilizing C. elegans as a model organism for toxicologic studies. Additionally, we observed altered non-mitochondrial respiration in two him strains, which may be of interest to other researchers utilizing these strains.
Conclusions
Basal mitochondrial parameters in male and hermaphrodite C. elegans are similar, at least at the whole-organism level, as is toxicity associated with a mitochondrial Complex I inhibitor, rotenone. Our data highlights the limitation of using C. elegans as a model to study sex-variable mitochondrial function and toxicological responses.
... The calculation of the respiration rates of the L-glutamine oxidation, utilizing Microsoft Excel version 16.78.3 software, was based on data interpretation by [22]. For the basal respiration rate, the third baseline measurement of the kinetic curve of OCR was used, as it was considered the most stable. ...
The neuroblastoma cell lines SH-SY5Y and Neuro2A are commonly utilized models in neurobiological research. DMEM supplemented with different nutrients and 5–10% Fetal Calf Serum (FCS) is typically used for culturing these cell lines. During special treatments, a reduced FCS content is often deployed to reduce cellular proliferation or the content of bioactive compounds. The impact of the reduction of FCS in culture media on the metabolic profile of SH-SY5Y and Neuro2A cells is currently unknown. Using an Amplex Red Assay, this study showed that the consumption of L-glutamine decreased after FCS reduction. Glucose and pyruvate consumption increased in both cell lines after the reduction of FCS. Thus, lactate production also increased with reduced FCS concentration. The reduction of FCS in the cell culture medium resulted in a reduced aerobic ATP production for SH-SY5Y cells and a complete shut down of aerobic ATP production for Neuro2A cells, measured using the Seahorse XF Real-Time ATP Rate Assay. Utilizing the Seahorse XF Glutamine Oxidation Stress Test, Neuro2A cells showed an increased utilization of L-glutamine oxidation after reduction of FCS. These results indicate that changes in FCS concentration in culture media have an impact on the different energy production strategies of SH-SY5Y and Neuro2A cells which must be considered when planning special treatments.
... Using the Mito Stress Test with XFe96, we assessed KPT-9274 impact on mitochondria function in 3D-cultured CP80 and ACI-98. As anticipated, KPT-9274 reduced oxygen consumption rate (OCR), an established measure of mitochondrial function [35], in CP80 and ACI-98 cells in 3D-spheroids, affecting both basal and maximal respiration (Fig. 3A, B). Interestingly, KPT-9274 significantly suppressed not only OCR, but also maximal extracellular acidification rate (ECAR), reflecting glycolysis (Fig. 3C). ...
Ovarian cancer follows a characteristic progression pattern, forming multiple tumor masses enriched with cancer stem cells (CSCs) within the abdomen. Most patients develop resistance to standard platinum-based drugs, necessitating better treatment approaches. Targeting CSCs by inhibiting NAD+ synthesis has been previously explored. Nicotinamide phosphoribosyltransferase (NAMPT), which is the rate limiting enzyme in the salvage pathway for NAD+ synthesis is an attractive drug target in this pathway. KPT-9274 is an innovative drug targeting both NAMPT and p21 activated kinase 4 (PAK4). However, its effectiveness against ovarian cancer has not been validated. Here, we show the efficacy and mechanisms of KPT-9274 in treating 3D-cultured spheroids that are resistant to platinum-based drugs. In these spheroids, KPT-9274 not only inhibited NAD+ production in NAMPT-dependent cell lines, but also suppressed NADPH and ATP production, indicating reduced mitochondrial function. It also downregulated of inflammation and DNA repair-related genes. Moreover, the compound reduced PAK4 activity by altering its mostly cytoplasmic localization, leading to NAD+-dependent decreases in phosphorylation of S6 Ribosomal protein, AKT, and β-Catenin in the cytoplasm. These findings suggest that KPT-9274 could be a promising treatment for ovarian cancer patients who are resistant to platinum drugs, emphasizing the need for precision medicine to identify the specific NAD+ producing pathway that a tumor relies upon before treatment.
... A total of 5 × 10 4 single cells were plated in their specific media (DMEM-F12, Gibco Invitrogen; supplemented with 10% FBS, 1/1000 pen/strep), and the assay was performed in XF seahorse DMEM medium supplemented with 25 mM glucose, 4 mM glutamine, and 5 mM pyruvate, pH 7.4. The optimal doses for each mitochondrial modulator were based on manufacturers' instructions and prior literature [19,29,30]: 1 µM oligomycin, 2 µM FCCP, and 1 µM Rot/AA (each), as these elicited an appropriate response in hCCs. ...
... Age-related modifications in the dynamic metabolic profile of hCCs were evaluated by comparing OCRs, a direct indicator of mitochondrial function. The OCR was monitored using extracellular flux analysis as described in other reproductive cell types [19,29,30]. Assessments were made at baseline (basal condition) and following the sequential addition of mitochondrial function modulators. ...
Age-related changes in the mitochondrial status of human cumulus cells (hCCs) impact oocyte quality; however, the relationship between hCC mitochondrial (dys)function and reproductive aging remains poorly understood. This study aimed to establish the interplay between hCC mitochondrial dysfunction and women’s reproductive potential. In this investigation, 266 women were enrolled and categorized into two groups based on their age: a young group (<35 years old) and an advanced maternal age (AMA) group (≥35 years old). Comprehensive analysis of reproductive outcomes was conducted in our population. Various mitochondrial-related parameters were analyzed across distinct subsets. Specifically, mitochondrial membrane potential (∆Ψm) and mitochondrial mass were examined in 53 samples, mtDNA content in 25 samples, protein levels in 23 samples, bioenergetic profiles using an XF24 Extracellular Flux Analyzer in 6 samples, and levels of reactive oxygen species (ROS) and adenosine triphosphate (ATP) in 39 and 43 samples, respectively. In our study, the reproductive potential of AMA women sharply decreased, as expected. Additionally, an impairment in the mitochondrial function of hCCs in older women was observed; however, no differences were found in terms of mitochondrial content. Regarding oxidative phosphorylation, metabolic profiling of hCCs from AMA women indicated a decrease in respiratory capacity, which was correlated with an age-dependent decrease in the ATP synthase (ATP5A1) protein level. However, intracellular ROS and ATP levels did not differ between groups. In conclusion, our study indicates that age-related dysfunction in hCCs is associated with impaired mitochondrial function, and, although further studies are required, ATP synthase could be relevant in this impairment.
... Importantly, this response is most likely driven by cumulus cells since oocytes cannot directly respond to FSH and have a limited capacity to metabolize glucose 23,47 . Additionally, a very low OCR rate was measured in denuded oocytes compared to intact COCs, reflecting the major contribution of cumulus cells 48 . ...
Oocyte maturation is a key process during which the female germ cell undergoes resumption of meiosis and completes its preparation for embryonic development including cytoplasmic and epigenetic maturation. The cumulus cells directly surrounding the oocyte are involved in this process by transferring essential metabolites, such as pyruvate, to the oocyte. This process is controlled by cyclic adenosine monophosphate (cAMP)-dependent mechanisms recruited downstream of follicle-stimulating hormone (FSH) signaling in cumulus cells. As mitochondria have a critical but poorly understood contribution to this process, we defined the effects of FSH and high cAMP concentrations on mitochondrial dynamics and function in porcine cumulus cells. During in vitro maturation (IVM) of cumulus-oocyte complexes (COCs), we observed an FSH-dependent mitochondrial elongation shortly after stimulation that led to mitochondrial fragmentation 24 h later. Importantly, mitochondrial elongation was accompanied by decreased mitochondrial activity and a switch to glycolysis. During a pre-IVM culture step increasing intracellular cAMP, mitochondrial fragmentation was prevented. Altogether, the results demonstrate that FSH triggers rapid changes in mitochondrial structure and function in COCs involving cAMP.
... Preparation and analysis were performed as in Muller et al. 71 . COC's (after 24 h IVM) were collected in Wash-TALP containing 0.3 mg/ml hyaluronidase to remove cumulus cells. ...
Maternal metabolic disorders may cause lipotoxic effects on the developing oocyte. Understanding the timing at which this might disrupt embryo epigenetic programming and how this is linked with mitochondrial dysfunction is crucial for improving assisted reproductive treatments, but has not been investigated before. Therefore, we used a bovine in vitro model to investigate if pathophysiological palmitic acid (PA) concentrations during in vitro oocyte maturation and in vitro embryo culture alter embryo epigenetic patterns (DNA methylation (5mC) and histone acetylation/methylation (H3K9ac/H3K9me2)) compared to control (CONT) and solvent control (SCONT), at the zygote and morula stage. Secondly, we investigated if these epigenetic alterations are associated with mitochondrial dysfunction and changes in ATP production rate, or altered expression of epigenetic regulatory genes. Compared to SCONT, H3K9ac and H3K9me2 levels were increased in PA-derived zygotes. Also, 5mC and H3K9me2 levels were increased in PA-exposed morulae compared to SCONT. This was associated with complete inhibition of glycolytic ATP production in oocytes, increased mitochondrial membrane potential and complete inhibition of glycolytic ATP production in 4-cell embryos and reduced SOD2 expression in PA-exposed zygotes and morulae. For the first time, epigenetic alterations in metabolically compromised zygotes and morulae have been observed in parallel with mitochondrial dysfunction in the same study.
... Even earlier, several specific mitochondrial features, including mitochondrial respiration, mitochondrial spare capacity (maximal mitochondrial respiration related to mitochondrial respiration) and efficiency of ATP generation (ATP-linked respiration related to mitochondrial respiration) were proposed for gaining a deeper understanding of mitochondrial function during embryo development in mice, rabbits 18,19 and the bovine 20,21 . Some of these mitochondrial parameters were recently shown to be determinable via an extracellular FLUX analyser 22 . However, no study on bovine embryos has been conducted taking advantage of this useful technical option of analysing oxygen consumption signatures of bovine embryos representing contrasting developmental capacities so far. ...
... Average OCR values per embryo measured within our study confirmed those observed in a previous study by Muller and collegues reporting values of 0.85 ± 0.08 pmol/min/embryo for in vitro derived bovine blastocysts 22 . ...
... for non-mitochondrial oxygen consumption, respectively, and thus did not differ significantly. This is within the range of a recent study of Muller and colleagues reporting a proportion of roughly 20% for non-mitochondrial respiration in in vitro derived embryos 22 . Also, VIVO, VITRO-HIGH and VITRO-LOW embryos utilized similar average amounts as well as relative proportions of total oxygen uptake for mitochondrial respiration in our study. ...
The major limitation of the widespread use of IVP derived embryos is their consistent deficiencies in vitality when compared with their ex vivo derived counterparts. Although embryo metabolism is considered a useful metric of embryo quality, research connecting mitochondrial function with the developmental capacity of embryos is still lacking. Therefore, the aim of the present study was to analyse bovine embryo respiration signatures in relation to developmental capacity. This was achieved by taking advantage of two generally accepted metrics for developmental capacity: (I) environmental conditions during development (vivo vs. vitro) and (II) developmental kinetics (day 7 vs. day 8 blastocysts). Our study showed that the developmental environment affected total embryo oxygen consumption while different morphokinetics illustrating the embryo qualities correlate with maximal mitochondrial respiration, mitochondrial spare capacity, ATP-linked respiration as well as efficiency of ATP generation. This respiration fingerprint for high embryo quality is reflected by relatively lower lipid contents and relatively higher ROS contents. In summary, the results of the present study extend the existing knowledge on the relationship between bovine embryo quality and the signature of mitochondrial respiration by considering contrasting developmental environments as well as different embryo morphokinetics.
... Using the Mito Stress Test with XFe96, we assessed KPT-9274 impact on mitochondria function in 3D-cultured CP80 and ACI-98. As anticipated, KPT-9274 reduced oxygen consumption rate (OCR), an established measure of mitochondrial function [35], in CP80 and ACI-98 cells in 3D spheroids, affecting both basal and maximal respiration ( Fig. 3A and 3B). Interestingly, KPT-9274 signi cantly suppressed not only OCR, but also maximal extracellular acidi cation rate (ECAR), re ecting glycolysis (Fig. 3C). ...
Ovarian cancer follows a characteristic progression pattern, forming multiple tumor masses enriched with cancer stem cells (CSCs) within the abdomen. Most patients develop resistance to standard platinum-based drugs, necessitating better treatment approaches. To target CSCs, inhibition of nicotinamide phosphoribosyltransferase (NAMPT), which is the rate-limiting enzyme in the salvage pathway for NAD ⁺ synthesis, has been explored in ovarian cancer. KPT-9274 is an innovative drug targeting both NAMPT and PAK4. However, its effectiveness against ovarian cancer has not been validated. Here, we show the efficacy and mechanisms of KPT-9274 in treating 3D-cultured spheroids that are resistant to platinum-based drugs. In these spheroids, KPT-9274 not only inhibited NAD ⁺ production in NAMPT-dependent cell lines, but also suppressed NADPH and ATP production, indicating reduced mitochondrial function. It also downregulated expression of genes involved with inflammation and DNA repair. Moreover, KPT-9274 impaired the kinase activity of PAK4 by altering the localization from cytoplasmic to nucleus, leading to decreased phosphorylation of S6 Ribosomal protein, AKT, and β-Catenin in the cytoplasm in a NAD ⁺ - dependent manner. These findings suggest that KPT-9274 is a promising treatment for ovarian cancer patients resistant to platinum drugs. Our study also indicates that the identification of specific NAD ⁺ -producing pathway is required for stratification of patients who can benefit from a KPT-9274 therapy.
