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Summary Mitochondrial membrane potential (ΔΨm) is an indicator of sperm quality and its evaluation complements the standard semen analysis. The fluorescent dye JC‐1 has been widely used to assess sperm ΔΨm; however, some problems have been detected under certain experimental conditions. Another fluorescent compound, tetramethylrhodamine methyl ester perchlorate (TMRM), has been used in somatic cells and bovine spermatozoa but not in human spermatozoa. TMRM accumulates in hyperpolarised mitochondria and the fluorescence intensity of this compound correlates with ΔΨm. Thus, the aim of this study was to evaluate and validate the usefulness of the fluorescent dye TMRM for measuring sperm ΔΨm. The results showed that TMRM accurately detects sperm populations displaying either high or low ΔΨm. Moreover, TMRM was able to measure sperm ΔΨm under the experimental conditions in which JC‐1 had previously presented difficulties. Differences in ΔΨm according to sperm and semen quality were properly detected and a positive correlation between ΔΨm and conventional semen parameters was observed. Finally, a positive correlation was found between the ΔΨm measurement by TMRM and by the widely used JC‐1. In conclusion, TMRM is a simple, time‐effective method, easy to set in laboratories equipped with flow cytometry technology, and can accurately detect changes in ΔΨm with efficiency comparable to JC‐1 without its limitations.

Ferroptosis is a recently recognized form of non‐apoptotic cell death caused by an iron‐dependent accumulation of lipid hydroperoxides, which plays important roles in a wide spectrum of pathological conditions. The present study was aimed to investigate the impact of ferroptosis on cisplatin‐induced sensory hair cell damage. Cell viability was determined by Cell Counting Kit‐8 and lactase dehydrogenase assays. The reactive oxygen species (ROS) levels were evaluated by 2,7‐Dichlorodi‐hydrofluorescein diacetate (DCFH‐DA) and MitoSox‐Red staining. Mitochondrial membrane potential (MMP) was measured by tetramethylrhodamine methyl ester (TMRM) staining. Lipid peroxidation, intracellular and mitochondrial iron were detected by Liperfluo, C11‐BODIPY581/591, FerroOrange and Mito‐FerroGreen, respectively. We found that cisplatin treatment not only markedly augmented ROS accumulation, decreased the MMP, but increased lipid peroxidation and iron accumulation in House Ear Institute‐Organ of Corti 1 (HEI‐OC1) cells. Of note, treatment with the specific ferroptosis inhibitor ferrostatin‐1 could effectively abrogate the cisplatin‐induced toxicity and subsequent cell death. Specifically, the improvement of mitochondrial functions is important mechanisms for protective action of ferroptosis inhibitor against cisplatin‐induced damages in HEI‐OC1 cells. Moreover, inhibition of ferroptosis significantly protected murine cochlear hair cells against cisplatin damage. In addition, treatment murine cochlear hair cells with ferroptosis inducer, RSL3, significantly exacerbated cisplatin‐induced damage, which could be alleviated by ROS inhibitor N‐acetyl‐L‐cysteine. Collectively, our study indicated that ferroptosis inhibition could alleviate the cisplatin‐induced ototoxicity via inactivation of lipid peroxide radical and improvement of mitochondrial function in hair cells.

In the ovarian follicle, a bilateral cell-to-cell communication exists between the female germ cell and the cumulus cells which surround the oocyte. This communication allows the transit of small size molecules known to impact oocyte developmental competence. Pyruvate derivatives produced by mitochondria, are one of these transferred molecules. Interestingly, mitochondria may adopt a variety of morphologies to regulate their functions. In this study, we described mitochondrial morphologies in porcine cumulus cells. Active mitochondria were stained with TMRM (Tetramethylrhodamine, Methyl Ester, Perchlorate) and observed with 2D confocal microscopy showing mitochondria of different morphologies such as short, intermediate, long, and very long. The number of mitochondria of each phenotype was quantified in cells and the results showed that most cells contained elongated mitochondria. Scanning electron microscopy (SEM) analysis confirmed at nanoscale resolution the different mitochondrial morphologies including round, short, intermediate, and long. Interestingly, 3D visualisation by focused ion-beam scanning electron microscopy (FIB-SEM) revealed different complex mitochondrial morphologies including connected clusters of different sizes, branched mitochondria, as well as individual mitochondria. Since mitochondrial dynamics is a key regulator of function, the description of the mitochondrial network organisation will allow to further study mitochondrial dynamics in cumulus cells in response to various conditions such as in vitro maturation.

Connexin43 (Cx43) exits as hemichannels in the inner mitochondrial membrane. We examined how mitochondrial Cx43 and mitochondrial KATP channels affect the occurrence of triggered arrhythmias. To generate cardiac-specific Cx43-deficient (cCx43−/−) mice, Cx43flox/flox mice were crossed with α-MHC (Myh6)-cre+/− mice. The resulting offspring, Cx43flox/flox/Myh6-cre+/− mice (cCx43−/− mice) and their littermates (cCx43+/+ mice), were used. Trabeculae were dissected from the right ventricles of mouse hearts. Cardiomyocytes were enzymatically isolated from the ventricles of mouse hearts. Force was measured with a strain gauge in trabeculae (22°C). To assess arrhythmia susceptibility, the minimal extracellular Ca2+ concentration ([Ca2+]o,min), at which arrhythmias were induced by electrical stimulation, was determined in trabeculae. ROS production was estimated with 2′,7′-dichlorofluorescein (DCF), mitochondrial membrane potential with tetramethylrhodamine methyl ester (TMRM), and Ca2+ spark frequency with fluo-4 and confocal microscopy in cardiomyocytes. ROS production within the mitochondria was estimated with MitoSoxRed and mitochondrial Ca2+ with rhod-2 in trabeculae. Diazoxide was used to activate mitochondrial KATP. Most of cCx43−/− mice died suddenly within 8 weeks. Cx43 was present in the inner mitochondrial membrane in cCx43+/+ mice but not in cCx43−/− mice. In cCx43−/− mice, the [Ca2+]o,min was lower, and Ca2+ spark frequency, the slope of DCF fluorescence intensity, MitoSoxRed fluorescence, and rhod-2 fluorescence were higher. TMRM fluorescence was more decreased in cCx43−/− mice. Most of these changes were suppressed by diazoxide. In addition, in cCx43−/− mice, antioxidant peptide SS-31 and N-acetyl-L-cysteine increased the [Ca2+]o,min. These results suggest that Cx43 deficiency activates Ca2+ leak from the SR, probably due to depolarization of mitochondrial membrane potential, an increase in mitochondrial Ca2+, and an increase in ROS production, thereby causing triggered arrhythmias, and that Cx43 hemichannel deficiency may be compensated by activation of mitochondrial KATP channels in mouse hearts.

Background The preference for glucose oxidative mode has crucial impacts on various physiological activities, including determining stem cell fate. External mechanical factors can play a decisive role in regulating critical metabolic enzymes and pathways of stem cells. Periodontal ligament stem cells (PDLSCs) are momentous effector cells that transform mechanical force into biological signals during the reconstruction of alveolar bone. However, mechanical stimuli-induced alteration of oxidative characteristics in PDLSCs and the underlying mechanisms have not been fully elucidated. Methods Herein, we examined the expression of LDH and COX4 by qRT-PCR, western blot, immunohistochemistry and immunofluorescence. We detected metabolites of lactic acid and reactive oxygen species for functional tests. We used tetramethylrhodamine methyl ester (TMRM) staining and a transmission electron microscope to clarify the mitochondrial status. After using western blot and immunofluorescence to clarify the change of DRP1, we further examined MFF, PINK1, and PARKIN by western blot. We used cyclosporin A (CsA) to confirm the regulation of mitophagy and ceased the stretching as a rescue experiment. Results Herein, we ascertained that mechanical force could increase the level of LDH and decrease the expression of COX4 in PDLSCs. Simultaneously, the yield of reactive oxygen species (ROS) in PDLSC reduced after stretching, while lactate acid augmented significantly. Furthermore, mitochondrial function in PDLSCs was negatively affected by impaired mitochondrial membrane potential (MMP) under mechanical force, and the augment of mitochondrial fission further induced PRKN-dependent mitophagy, which was confirmed by the rescue experiments via blocking mitophagy. As a reversible physiological stimulation, the anaerobic preference of PDLSCs altered by mechanical force could restore after the cessation of force stimulation. Conclusions Altogether, our study demonstrates that PDLSCs under mechanical force preferred anaerobic oxidation induced by the affected mitochondrial dynamics, especially mitophagy. Our findings support an association between mechanical stimulation and the oxidative profile of stem cells, which may shed light on the mechanical guidance of stem cell maintenance and commitment, and lay a molecular foundation for periodontal tissue regeneration. Graphical Abstract

Iron (Fe) plays an essential role in many physiological processes. Hereditary hemochromatosis or frequent blood transfusions often cause iron overload (IO), which can lead to cardiomyopathy and arrhythmias; however, the underlying mechanism is not well defined. In the present study, we assess the hypothesis that IO promotes arrhythmias via reactive oxygen species (ROS) production, mitochondrial membrane potential (∆Ψm) depolarization, and disruption of cytosolic Ca dynamics. In ventricular myocytes isolated from wild type (WT) mice, both cytosolic and mitochondrial Fe levels were elevated following perfusion with the Fe3+/8-hydroxyquinoline (8-HQ) complex. IO promoted mitochondrial superoxide generation (measured using MitoSOX Red) and induced the depolarization of the ΔΨm (measured using tetramethylrhodamine methyl ester, TMRM) in a dose-dependent manner. IO significantly increased the rate of Ca wave (CaW) formation measured in isolated ventricular myocytes using Fluo-4. Furthermore, in ex-vivo Langendorff-perfused hearts, IO increased arrhythmia scores as evaluated by ECG recordings under programmed S1-S2 stimulation protocols. We also carried out similar experiments in cyclophilin D knockout (CypD KO) mice in which the mitochondrial permeability transition pore (mPTP) opening is impaired. While comparable cytosolic and mitochondrial Fe load, mitochondrial ROS production, and depolarization of the ∆Ψm were observed in ventricular myocytes isolated from both WT and CypD KO mice, the rate of CaW formation in isolated cells and the arrhythmia scores in ex-vivo hearts were significantly lower in CypD KO mice compared to those observed in WT mice under conditions of IO. The mPTP inhibitor cyclosporine A (CsA, 1 µM) also exhibited a protective effect. In conclusion, our results suggest that IO induces mitochondrial ROS generation and ∆Ψm depolarization, thus opening the mPTP, thereby promoting CaWs and cardiac arrhythmias. Conversely, the inhibition of mPTP ameliorates the proarrhythmic effects of IO.

The culture of embryos in the non-essential amino acid L-proline (Pro) or its analogues pipecolic acid (PA) and L-4-thiazolidine carboxylic acid (L4T) improves embryo development, increasing the percentage that develop to the blastocyst stage and hatch. Staining of 2-cell and 4-cell embryos with tetramethylrhodamine methyl ester and 2′,7′-dichlorofluorescein diacetate showed that the culture of embryos in the presence of Pro, or either of these analogues, reduced mitochondrial activity and reactive oxygen species (ROS), respectively, indicating potential mechanisms by which embryo development is improved. Inhibition of the Pro metabolism enzyme, proline oxidase, by tetrahydro-2-furoic-acid prevented these reductions and concomitantly prevented the improved development. The ways in which Pro, PA and L4T reduce mitochondrial activity and ROS appear to differ, despite their structural similarity. Specifically, the results are consistent with Pro reducing ROS by reducing mitochondrial activity while PA and L4T may be acting as ROS scavengers. All three may work to reduce ROS by contributing to the GSH pool. Overall, our results indicate that reduction in mitochondrial activity and oxidative stress are potential mechanisms by which Pro and its analogues act to improve pre-implantation embryo development.

SummaryBreast cancer is the leading cause of cancer death among women worldwide. For this reason, the development of new therapies is still essential. In this work we have analyzed the antitumor potential of levoglucosenone, a chiral building block derived from the pyrolysis of cellulose-containing materials such as soybean hulls, and three structurally related analogues. Employing human and murine mammary cancer models, we have evaluated the effect of our compounds on cell viability through MTS assay, apoptosis induction by acridine orange/ethidium bromide staining and/or flow cytometry and the loss of mitochondrial potential by tetramethylrhodamine methyl ester staining. Autophagy and senescence induction were also evaluated by Western blot and β-galactosidase activity respectively. Secreted metalloproteases activity was determined by quantitative zymography. Migratory capacity was assessed by wound healing assays while invasive potential was analyzed using Matrigel-coated transwell chambers. In vivo studies were also performed to evaluate subcutaneous tumor growth and experimental lung colonization. All compounds impaired in vitro proliferation with IC50 values in a range of low micromolar. Apoptosis was identified as the main mechanism responsible for the reduction of monolayer cell content induced by the compounds without detecting modulations of autophagy or senescence processes. Two of the four compounds (levoglucosenone and its brominated variant) were able to modulate in vitro events associated with tumor progression, such as migratory potential, invasiveness, and proteases secretion. Furthermore, tumor volume and metastatic spread were significantly reduced in vivo after the treatment these two compounds. Here, we could obtain from soybean hulls, a material with almost no commercial value, a variety of chemical compounds useful for breast cancer treatment.

The MTT assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in the cell cytoplasm to a strongly light absorbing formazan is among the most commonly used methods for determination of cell viability and activity of NAD-dependent oxidoreductases. In the present study, the effects of MTT (0.1 mg/ml) on mitochondrial potential (ΔΨ m ), intracellular NADH, and respiration of cultured rat cerebellum neurons and isolated rat liver mitochondria were investigated. MTT caused rapid quenching of NADH autofluorescence, fluorescence of MitoTracker Green (MTG) and ΔΨ m -sensitive probes Rh123 (rhodamine 123) and TMRM (tetramethylrhodamine methyl ester). The Rh123 signal, unlike that of NADH, MTG, and TMRM, increased in the nucleoplasm after 5-10 min, and this was accompanied by the formation of opaque aggregates of formazan in the cytoplasm and neurites. Increase in the Rh123 signal indicated diffusion of the probe from mitochondria to cytosol and nucleus due to ΔΨ m decrease. Inhibition of complex I of the respiratory chain decreased the rate of formazan formation, while inhibition of complex IV increased it. Inhibition of complex III and ATP-synthase affected only insignificantly the rate of formazan formation. Inhibition of glycolysis by 2-deoxy-D-glucose blocked the MTT reduction, whereas pyruvate increased the rate of formazan formation in a concentration-dependent manner. MTT reduced the rate of oxygen consumption by cultured neurons to the value observed when respiratory chain complexes I and III were simultaneously blocked, and it suppressed respiration of isolated mitochondria if substrates oxidized by NAD-dependent dehydrogenases were used. These results demonstrate that formazan formation in cultured rat cerebellum neurons occurs primarily in mitochondria. The initial rate of formazan formation may serve as an indicator of complex I activity and pyruvate transport rate.

To investigate the effect of Da-Bu-Yin-Wan and Qian-Zheng-San (DBYW and QZS) on mitochondrial mass in Parkinson's disease (PD) cell model induced by 1-Methyl-4-phenylpyridinium Ion (MPP ). The SH-SY5Y cell was selected and treated with MPP . The PD model was intervened with DBYW and QZS. CCK-8 method was used to detect the survival rate of cells in each group. Mitochondria was labeled by mitoTracker Red CMXRos probe and observed by laser scanning confocal microscope, and ImageJ software was used to process images and measure mitochondrial form factors; Tetramethylrhodamine methyl ester was used to detect mitochondrial membrane potential (ΔΨm); Luciferase method was used to detect cellular ATP levels; Western-Blot technique was applied to detect the expression levels of Parkin protein, and the expression levels of Mfn1, Mfn2, OPA1, Drp1, and Fis1. We found that DBYW and QZS treatment significantly increased the cell survival rate, form factor (F-factor), mitochondrial activity and ΔΨm after MPP treatment, while the increase of ATP levels was not significant. In addition, the results of western blot analysis showed that the MPP induced increase in the expression of Drp1 and Fis1, as well as decrease in Parkin, Mfn1, Mfn2, and OPA1 were all partially revised by DBYW and QZS. In summary, our data strongly suggested that DBYW and QZS treatment can exert protective effects against PD related neuronal injury through regulation the homeostasis between mitochondrial fission and fusion.