Protein-mediated energy-dissipating pathways in mitochondria

Mitochondrial production of reactive oxygen species (ROS) is a well-established fact of fundamental importance to aging and etiology of many pathologies with serious public health implications. The ROS production is an innate property of mitochondrial biochemistry inseparable from the oxidative metabolism. Recent discoveries indicate that in addition to several ROS-detoxifying enzyme systems, which remove ROS, mitochondria may also be able to limit their ROS production by the mechanism comprising several protein-mediated energy-dissipating ("uncoupling") pathways. Although the physiological significance and in vivo modus operandi of these pathways remain to be elucidated, several proteins potentially capable of energy dissipation are known. This mini-review addresses the identity of mitochondrial protein-mediated energy-dissipating pathways and the experimental evidence to their role in controlling ROS production.     

Possible plant mitochondria involvement in cell adaptation to drought stress. A case study: durum wheat mitochondria

Although plant cell bioenergetics is strongly affected by abiotic stresses, mitochondrial metabolism under stress is still largely unknown. Interestingly, plant mitochondria may control reactive oxygen species (ROS) generation by means of energy-dissipating systems. Therefore, mitochondria may play a central role in cell adaptation to abiotic stresses, which are known to induce oxidative stress at cellular level. With this in mind, in recent years, studies have been focused on mitochondria from durum wheat, a species well adapted to drought stress. Durum wheat mitochondria possess three energy-dissipating systems: the ATP-sensitive plant mitochondrial potassium channel (PmitoK(ATP)); the plant uncoupling protein (PUCP); and the alternative oxidase (AOX). It has been shown that these systems are able to dampen mitochondrial ROS production; surprisingly, PmitoK(ATP) and PUCP (but not AOX) are activated by ROS. This was found to occur in mitochondria from both control and hyperosmotic-stressed seedlings. Therefore, the hypothesis of a 'feed-back' mechanism operating under hyperosmotic/oxidative stress conditions was validated: stress conditions induce an increase in mitochondrial ROS production; ROS activate PmitoK(ATP) and PUCP that, in turn, dissipate the mitochondrial membrane potential, thus inhibiting further large-scale ROS production. Another important aspect is the chloroplast/cytosol/mitochondrion co-operation in green tissues under stress conditions aimed at modulating cell redox homeostasis. Durum wheat mitochondria may act against chloroplast/cytosol over-reduction: the malate/oxaloacetate antiporter and the rotenone-insensitive external NAD(P)H dehydrogenases allow cytosolic NAD(P)H oxidation; under stress this may occur without high ROS production due to co-operation with AOX, which is activated by intermediates of the photorespiratory cycle.     

Reactive oxygen species generation and antioxidant systems in plant mitochondria

In living cells, reactive oxygen species (ROS) play a key role in signaling but these compounds can also damage macromolecules. As in other compartments, the mitochondrial ROS concentrations need to be tightly controlled. Plant mitochondria contain several antioxidant systems that are not only able to scavenge ROS and limit their production but also to repair damages to macromolecules and possibly to serve as redox sensors. They comprise ascorbate- and glutathione-dependent pathways as well as systems based on thioredoxin (TRX)- and glutaredoxin (GRX)-like molecules. This review describes the various mitochondrial redox pathways for ROS control in plants with special emphasis on the poorly studied GRX and TRX systems and provides perspectives for future research in this area.     

Mitochondrial energy-dissipating systems (alternative oxidase,uncoupling proteins,and external NADH dehydrogenase) are involved in development of frost-resistance of winter wheat seedlings

Gene expression, protein synthesis, and activities of alternative oxidase (AOX), uncoupling proteins (UCP), adenine nucleotide translocator (ANT), and non-coupled NAD(P)H dehydrogenases (NDex, NDPex, and NDin) were studied in shoots of etiolated winter wheat (Triticum aestivum L.) seedlings after exposure to hardening low positive (2°C for 7 days) and freezing (?2°C for 2 days) temperatures. The cold hardening efficiently increased frost-resistance of the seedlings and decreased the generation of reactive oxygen species (ROS) during further cold shock. Functioning of mitochondrial energy-dissipating systems can represent a mechanism responsible for the decrease in ROS under these conditions. These systems are different in their response to the action of the hardening low positive and freezing temperatures. The functioning of the first system causes induction of AOX and UCP synthesis associated with an increase in electron transfer via AOX in the mitochondrial respiratory chain and also with an increase in the sensitivity of mitochondrial non-phosphorylating respiration to linoleic and palmitic acids. The increase in electron transfer via AOX upon exposure of seedlings to hardening freezing temperature is associated with retention of a high activity of NDex. It seems that NDex but not the NDPex and NDin can play an important role in maintaining the functional state of mitochondria in heterotrophic tissues of plants under the influence of freezing temperatures. The involvement of the mitochondrial energy-dissipating systems and their possible physiological role in the adaptation of winter crops to cold and frost are discussed.     

The role of mitochondria in protection of the heart by preconditioning

A prolonged period of ischaemia followed by reperfusion irreversibly damages the heart. Such reperfusion injury (RI) involves opening of the mitochondrial permeability transition pore (MPTP) under the conditions of calcium overload and oxidative stress that accompany reperfusion. Protection from MPTP opening and hence RI can be mediated by ischaemic preconditioning (IP) where the prolonged ischaemic period is preceded by one or more brief (2-5 min) cycles of ischaemia and reperfusion. Following a brief overview of the molecular characterisation and regulation of the MPTP, the proposed mechanisms by which IP reduces pore opening are reviewed including the potential roles for reactive oxygen species (ROS), protein kinase cascades, and mitochondrial potassium channels. It is proposed that IP-mediated inhibition of MPTP opening at reperfusion does not involve direct phosphorylation of mitochondrial proteins, but rather reflects diminished oxidative stress during prolonged ischaemia and reperfusion. This causes less oxidation of critical thiol groups on the MPTP that are known to sensitise pore opening to calcium. The mechanisms by which ROS levels are decreased in the IP hearts during prolonged ischaemia and reperfusion are not known, but appear to require activation of protein kinase Cepsilon, either by receptor-mediated events or through transient increases in ROS during the IP protocol. Other signalling pathways may show cross-talk with this primary mechanism, but we suggest that a role for mitochondrial potassium channels is unlikely. The evidence for their activity in isolated mitochondria and cardiac myocytes is reviewed and the lack of specificity of the pharmacological agents used to implicate them in IP is noted. Some K(+) channel openers uncouple mitochondria and others inhibit respiratory chain complexes, and their ability to produce ROS and precondition hearts is mimicked by bona fide uncouplers and respiratory chain inhibitors. IP may also provide continuing protection during reperfusion by preventing a cascade of MPTP-induced ROS production followed by further MPTP opening. This phase of protection may involve survival kinase pathways such as Akt and glycogen synthase kinase 3 (GSK3) either increasing ROS removal or reducing mitochondrial ROS production.     

The role of mitochondria in protection of the heart by preconditioning

A prolonged period of ischaemia followed by reperfusion irreversibly damages the heart. Such reperfusion injury (RI) involves opening of the mitochondrial permeability transition pore (MPTP) under the conditions of calcium overload and oxidative stress that accompany reperfusion. Protection from MPTP opening and hence RI can be mediated by ischaemic preconditioning (IP) where the prolonged ischaemic period is preceded by one or more brief (2-5 min) cycles of ischaemia and reperfusion. Following a brief overview of the molecular characterisation and regulation of the MPTP, the proposed mechanisms by which IP reduces pore opening are reviewed including the potential roles for reactive oxygen species (ROS), protein kinase cascades, and mitochondrial potassium channels. It is proposed that IP-mediated inhibition of MPTP opening at reperfusion does not involve direct phosphorylation of mitochondrial proteins, but rather reflects diminished oxidative stress during prolonged ischaemia and reperfusion. This causes less oxidation of critical thiol groups on the MPTP that are known to sensitise pore opening to calcium. The mechanisms by which ROS levels are decreased in the IP hearts during prolonged ischaemia and reperfusion are not known, but appear to require activation of protein kinase Cε, either by receptor-mediated events or through transient increases in ROS during the IP protocol. Other signalling pathways may show cross-talk with this primary mechanism, but we suggest that a role for mitochondrial potassium channels is unlikely. The evidence for their activity in isolated mitochondria and cardiac myocytes is reviewed and the lack of specificity of the pharmacological agents used to implicate them in IP is noted. Some K⁺ channel openers uncouple mitochondria and others inhibit respiratory chain complexes, and their ability to produce ROS and precondition hearts is mimicked by bona fide uncouplers and respiratory chain inhibitors. IP may also provide continuing protection during reperfusion by preventing a cascade of MPTP-induced ROS production followed by further MPTP opening. This phase of protection may involve survival kinase pathways such as Akt and glycogen synthase kinase 3 (GSK3) either increasing ROS removal or reducing mitochondrial ROS production.     

Mitochondrial metabolism of reactive oxygen species

For a long time mitochondria have mainly been considered for their role in the aerobic energy production in eukaryotic cells, being the sites of the oxidative phosphorylation, which couples the electron transfer from respiratory substrates to oxygen with the ATP synthesis. Subsequently, it was showed that electron transfer along mitochondrial respiratory chain also leads to the formation of radicals and other reactive oxygen species, commonly indicated as ROS. The finding that such species are able to damage cellular components, suggested mitochondrial involvement in degenerative processes underlying several diseases and aging.More recently, a new role for mitochondria, as a system able to supply protection against cellular oxidative damage, is emerging. Experimental evidence indicates that the systems, evolved to protect mitochondria against endogenously produced ROS, can also scavenge ROS produced by other cellular sources. It is possible that this action, particularly relevant in physio-pathological conditions leading to increased cellular ROS production, is more effective in tissues provided with abundant mitochondrial population. Moreover, the mitochondrial dysfunction, resulting from ROS-induced inactivation of important mitochondrial components, can be attenuated by the cell purification from old ROS-overproducing mitochondria, which are characterized by high susceptibility to oxidative damage. Such an elimination is likely due to two sequential processes, named mitoptosis and mitophagy, which are usually believed to be induced by enhanced mitochondrial ROS generation. However, they could also be elicited by great amounts of ROS produced by other cellular sources and diffusing into mitochondria, leading to the elimination of the old dysfunctional mitochondrial subpopulation.     

Regulation of necrosis of H9c2 myogenic cells upon transient energy deprivation. Rapid deenergization of mitochondria precedes necrosis and is controlled by reactive oxygen species, stress kinase JNK, HSP72 and ARC

Subjecting myogenic H9c2 cells to transient energy deprivation leads to a caspase-independent death with typical features of necrosis. Here we show that the rupture of cytoplasmic membrane, the terminal event in necrosis, is shortly preceded by rapid depolarization of mitochondrial membranes. The rapid deenergization of mitochondria critically depended upon prior generation of reactive oxygen species (ROS) during ATP depletion stage. Accordingly, expression of catalase prevented mitochondrial depolarization and averted subsequent necrosis. Interestingly, trifluoperazine, a compound that protects cells from ischemic insults, prevented necrosis of H9c2 cells through inhibition of ROS production. Other factors that regulated the mitochondrial membrane depolarization and subsequent loss of plasma membrane integrity include a stress kinase JNK activated at early steps of recovery from ATP depletion, as well as an apoptotic inhibitory protein ARC. Accordingly, inhibition of JNK or overexpression of ARC prevented mitochondrial depolarization and rescued H9c2 cells from necrosis. ROS and JNK affected mitochondrial deenergization and necrosis independently of each other since inhibition of ROS production did not prevent activation of JNK, whereas inhibition of JNK did not suppress ROS accumulation. Therefore, JNK activation and ROS production represent two independent pathways that control mitochondrial depolarization and subsequent necrosis of cells subjected to transient energy deprivation. Overexpression of ARC, although preventing mitochondrial depolarization, did not affect either JNK activation or production of ROS. The major heat shock protein Hsp72 inhibited JNK-related steps of necrotic pathway but did not affect ROS accumulation. Interestingly, mitochondrial depolarization and subsequent necrosis can be suppressed by an Hsp72 mutant Hsp72DeltaEEVD, which lacks chaperone function but can efficiently suppress JNK activation. Thus, Hsp72 is directly implicated in a signaling pathway, which leads to necrotic death.     

Reactive oxygen species mediated placental oxidative stress,mitochondrial content,and cell cycle progression through mitogen-activated protein kinases in intrauterine growth restricted pigs

Intrauterine growth restriction (IUGR) remains a significant obstacle in pig production; however, information regarding the relationship between reactive oxygen species (ROS)-induced placental dysfunction and IUGR is still unknown. This study aimed to explore the placental redox status, mitochondrial content, cellular progression, and mitogen-activated protein kinase (MAPK) pathways in IUGR. Placental tissues were collected from normal intrauterine gestation (NIUG) and IUGR fetuses at delivery. Compared with the NIUG, placental ROS production, lipid peroxidation, and DNA damage were increased in IUGR. Placental mitochondrial DNA (mtDNA) content and mtDNA-encoded gene expression decreased in IUGR. Moreover, p21 phosphorylation increased, cyclin E expression decreased in IUGR cases, which showed senescence characteristics. Analysis of signaling pathways showed that the ERK1/2 phosphorylation increased whereas the p38 and JNK phosphorylation decreased in IUGR. In cultured porcine trophectoderm (pTr) cells, exogenous H₂O₂ increased intracellular ROS production, decreased cell viability in a dose-dependent manner. Cell cycle distribution was found to arrest in S and G2/M phases. Our findings suggested that IUGR was associated with greater placental ROS and oxidative injury, which might be a factor that resulted in lower mitochondrial content, microvilli loss and senescence, and activation of MAPK pathways.     

Constitutive Reactive Oxygen Species Generation from Autophagosome/Lysosome in Neuronal Oxidative Toxicity

Reactive oxygen species (ROS) are involved in several cell death processes, including cerebral ischemic injury. We found that glutamate-induced ROS accumulation and the associated cell death in mouse hippocampal cell lines were delayed by pharmacological inhibition of autophagy or lysosomal activity. Glutamate, however, did not stimulate autophagy, which was assessed by a protein marker, LC3, and neither changes in organization of mitochondria nor lysosomal membrane permeabilization were observed. Fluorescent analyses by a redox probe PF-H₂TMRos revealed that autophagosomes and/or lysosomes are the major sites for basal ROS generation in addition to mitochondria. Treatments with inhibitors for autophagy and lysosomes decreased their basal ROS production and caused a burst of mitochondrial ROS to be delayed. On the other hand, attenuation of mitochondrial activity by serum depletion or by high cell density culture resulted in the loss of both constitutive ROS production and an ROS burst in mitochondria. Thus, constitutive ROS production within mitochondria and lysosomes enables cells to be susceptible to glutamate-induced oxidative cytotoxicity. Likewise, inhibitors for autophagy and lysosomes reduced neural cell death in an ischemia model in rats. We suggest that cell injury during periods of ischemia is regulated by ROS-generating activity in autophagosomes and/or lysosomes as well as in mitochondria.     

Reactive oxygen species and hormonal control of cell death

The accumulation of reactive oxygen species (ROS) is involved in regulating cell death. Pathogen- and ozone-induced processes have become important models for the study of cell death regulation by ROS. Hydrogen peroxide and superoxide have emerged as the two key ROS and recent studies have addressed their sources and control of their production. ROS signals interact directly or indirectly with several other signaling pathways, such as nitric oxide, and the stress hormones salicylic acid, jasmonic acid and ethylene. The interaction and balance of these pathways determines whether the cell lives or dies.     

Mitochondrial metabolism of reactive oxygen species

Oxidative stress is considered a major contributor to etiology of both normal senescence and severe pathologies with serious public health implications. Mitochondria generate reactive oxygen species (ROS) that are thought to augment intracellular oxidative stress. Mitochondria possess at least nine known sites that are capable of generating superoxide anion, a progenitor ROS. Mitochondria also possess numerous ROS defense systems that are much less studied. Studies of the last three decades shed light on many important mechanistic details of mitochondrial ROS production, but the bigger picture remains obscure. This review summarizes the current knowledge about major components involved in mitochondrial ROS metabolism and factors that regulate ROS generation and removal. An integrative, systemic approach is applied to analysis of mitochondrial ROS metabolism, which is now dissected into mitochondrial ROS production, mitochondrial ROS removal, and mitochondrial ROS emission. It is suggested that mitochondria augment intracellular oxidative stress due primarily to failure of their ROS removal systems, whereas the role of mitochondrial ROS emission is yet to be determined and a net increase in mitochondrial ROS production in situ remains to be demonstrated.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 246–264.Original Russian Text Copyright © 2005 by Andreyev, Kushnareva, Starkov.This revised version was published online in April 2005 with corrections to the post codes.     

Mitochondrial NADH redox potential impacts the reactive oxygen species production of reverse Electron transfer through complex I

There is substantial evidence that Reactive Oxygen Species (ROS) play a major part in cell functioning. Although their harmfulness through oxidative stress is well documented, their role in signaling and sensing as an oxidative signal still needs to be investigated. In most cells, the mitochondrial Electron Transport Chain (ETC) is the primary source of ROS. The production of ROS by reverse electron transfer through complex I has been demonstrated both in an experimental context but also in many pathophysiological situations. Therefore, understanding the mechanisms that regulate this ROS production is of great interest to control its harmful effects. We used nigericin, Pi and valinomycin as tools to modulate the pH gradient (?pH) and the membrane potential (?Ψ) of the protonmotive force (?p) in liver and muscle mitochondria to accurately determine how these parameters control the ROS production. We show that a high ?Ψ is the “sine qua none” condition for ROS production from the reverse electron transfer (RET) through the complex I. However, a high ?Ψ is not the only condition governing ROS production. Indeed, using tools that modulate the mitochondrial NADH level, we also demonstrate that ROS production is directly related to the mitochondrial redox potential when the membrane potential is almost stable.     

ROS production by adrenodoxin does not cause apoptosis in fission yeast

We previously showed that production of reactive oxygen species (ROS) caused by overexpression of the mitochondrial electron transfer protein adrenodoxin (Adx) induces apoptosis in mammalian cells. In the fission yeast Schizosaccharomyces pombe, ROS are also produced in cells that undergo an apoptotic-like cell death, but it is not yet clear whether they are actually causative for this phenomenon or whether they are merely produced as a by-product. Therefore, the purpose of this study was to trigger mitochondrial ROS production in fission yeast by overexpression of either wildtype Adx (Adx-WT) or of several activated Adx mutants and to investigate its consequences. It was found that strong expression of either Adx-WT or Adx-S112W did not produce any ROS, while Adx-D113Y caused a twofold and Adx^1–108 a threefold increase in ROS formation as compared to basal levels. However, no typical apoptotic markers or decreased viability could be observed in these strains. Since we previously observed that an increase in mitochondrial ROS formation of about 60% above basal levels is sufficient to strongly induce apoptosis in mammalian cells, we conclude that S. pombe is either very robust to mitochondrial ROS production or does not undergo apoptotic cell death in response to mitochondrial ROS at all.     

Mitochondria in exercise-induced oxidative stress

In recent years it has been suggested that reactive oxygen species (ROS) are involved in the damage to muscle and other tissues induced by acute exercise. Despite the small availability of direct evidence for ROS production during exercise, there is an abundance of literature providing indirect support that oxidative stress occurs during exercise. The electron transport associated with the mitochondrial respiratory chain is considered the major process leading to ROS production at rest and during exercise. It is widely assumed that during exercise the increased electron flow through the mitochondrial electron transport chain leads to an increased rate of ROS production. On the other hand, results obtained by in vitro experiments indicate that mitochondrial ROS production is lower in state 3 (ADP-stimulated) than in state 4 (basal) respiration. It is possible, however, that factors, such as temperature, that are modified in vivo during intense physical activity induce changes (uncoupling associated with loss of cytochrome oxidase activity) leading to increased ROS production. The mitochondrial respiratory chain could also be a potential source of ROS in tissues, such as liver, kidney and nonworking muscles, that during exercise undergo partial ischemia because of reduced blood supply. Sufficient oxygen is available to interact with the increasingly reduced respiratory chain and enhance the ROS generation. At the cessation of exercise, blood flow to hypoxic tissues resumes leading to their reoxygenation. This mimics the ischemia-reperfusion phenomenon, which is known to cause excessive production of free radicals. Apart from a theoretical rise in ROS, there is little evidence that exercise-induced oxidative stress is due to its increased mitochondrial generation. On the other hand, if mitochondrial production of ROS supplies a remarkable contribution to exercise-induced oxidative stress, mitochondria should be a primary target of oxidative damage. Unfortunately, there are controversial reports concerning the exercise effects on structural and functional characteristics of mitochondria. However, the isolation of mitochondrial fractions by differential centrifugation has shown that the amount of damaged mitochondria, recovered in the lightest fraction, is remarkably increased by long-lasting exercise.     

Mitochondria contribute to LPS-induced MAPK activation via uncoupling protein UCP2 in macrophages

The mitochondrion is a major organelle contributing to energy metabolism but also a main site of ROS (reactive oxygen species) production. LPS (lipopolysaccharide)-induced ROS signalling is a critical event in macrophage activation. In the present paper we report that part of LPS-mediated ROS signalling comes from mitochondria inside a signal amplification loop that enhances MAPK (mitogen-activated protein kinase) activation. More precisely, we have identified the inner mitochondrial membrane UCP2 (uncoupling protein 2) as a physiological brake on ROS signalling. Stimulation of murine bone marrow-derived macrophages by LPS quickly down-regulated UCP2 through the JNK (c-Jun N-terminal kinase) and p38 pathways. UCP2 down-regulation was shown to be necessary to increase mitochondrial ROS production in order to potentiate MAPK activation. Consistent with this, UCP2-deficient macrophages exhibit an enhanced inflammatory state characterized by increased nitric oxide production and elevated migration ability. Additionally, we found that the absence of UCP2 renders macrophages more resistant to nitric oxide-induced apoptosis.     

Mitochondrial Ca2+-activated K+ channels and their role in cell life and death pathways

Ca²⁺-activated K⁺ channels (K_Ca) are expressed at the plasma membrane and in cellular organelles. Expression of all K_Ca channel subtypes (BK, IK and SK) has been detected at the inner mitochondrial membrane of several cell types. Primary functions of these mitochondrial K_Ca channels include the regulation of mitochondrial ROS production, maintenance of the mitochondrial membrane potential and preservation of mitochondrial calcium homeostasis. These channels are therefore thought to contribute to cellular protection against oxidative stress through mitochondrial mechanisms of preconditioning. In this review, we summarize the current knowledge on mitochondrial KCa channels, and their role in mitochondrial function in relation to cell death and survival pathways. More specifically, we systematically discuss studies on the role of these mitochondrial K_Ca channels in pharmacological preconditioning, and according protective effects on ischemic insults to the brain and the heart.     

Importance of mitochondria in survival of Cryptococcus neoformans under low oxygen conditions and tolerance to cobalt chloride

Cryptococcus neoformans is an environmental fungal pathogen that requires atmospheric levels of oxygen for optimal growth. For the fungus to be able to establish an infection, it must adapt to the low oxygen concentrations in the host environment compared to its natural habitat. In order to investigate the oxygen sensing mechanism in C. neoformans, we screened T-DNA insertional mutants for hypoxia-mimetic cobalt chloride (CoCl(2))-sensitive mutants. All the CoCl(2)-sensitive mutants had a growth defect under low oxygen conditions at 37 degrees C. The majority of mutants are compromised in their mitochondrial function, which is reflected by their reduced rate of respiration. Some of the mutants are also defective in mitochondrial membrane permeability, suggesting the importance of an intact respiratory system for survival under both high concentrations of CoCl(2) as well as low oxygen conditions. In addition, the mutants tend to accumulate intracellular reactive oxygen species (ROS), and all mutants show sensitivity to various ROS generating chemicals. Gene expression analysis revealed the involvement of several pathways in response to cobalt chloride. Our findings indicate cobalt chloride sensitivity and/or sensitivity to low oxygen conditions are linked to mitochondrial function, sterol and iron homeostasis, ubiquitination, and the ability of cells to respond to ROS. These findings imply that multiple pathways are involved in oxygen sensing in C. neoformans.     

Inhibition of ROS production through mitochondria-targeted antioxidant and mitochondrial uncoupling increases post-thaw sperm viability in yellow catfish

Reactive oxygen species (ROS) are one of the main causes for decreased viability in cryopreserved sperm. Many studies have reported the beneficial effect of antioxidant supplements in freezing media for post-thaw sperm quality. In the present study, we explored two new approaches of ROS inhibition in sperm cryopreservation of yellow catfish, namely mitochondrial-targeted antioxidant and metabolic modulator targeting mitochondrial uncoupling pathways. Our study revealed that addition of MitoQ, a compound designed to deliver ubiquinone into mitochondria, significantly decreased ROS production, as well as lipid peroxidation, and increased post-thaw viability. Similarly, sperm incubated with 2,4-dinitrophenol (DNP), a chemical protonophore that induces mitochondrial uncoupling, also had reduced ROS production, as well as lipid peroxidation, and increased post-thaw sperm viability. Conversely, activation of uncoupling protein (UCP2) by 4-hydroxynonenal (HNE) neither reduced ROS production nor increased post-thaw sperm viability. Our findings indicate that ROS inhibition through mitochondrial-targeted antioxidant or mild mitochondrial uncoupling is beneficial for sperm cryopreservation in yellow catfish. Our study provides novel methods to mitigate oxidative stress induced damage in cryopreserved sperm for future applications.