Reduced metabolic rate and oxygen radicals production in stored insect sperm

International audienceAbstractFemales of internally fertilizing species can significantly extend sperm lifespan and functionality during sperm storage. The mechanisms for such delayed cellular senescence remain unknown. Here, we apply current hypotheses of cellular senescence developed for diploid cells to sperm cells, and empirically test opposing predictions on the relationship between sperm metabolic rate and oxygen radical production in an insect model, the cricket Gryllus bimaculatus. Using time-resolved microfluorimetry, we found a negative correlation between metabolic rate (proportion of protein-bound NAD[P]H) and in situ intracellular oxygen radicals production in freshly ejaculated sperm. In contrast, sperm stored by females for periods of 1 h to 26 days showed a positive correlation between metabolic rate and oxygen radicals production. At the same time, stored sperm showed a 37 per cent reduced metabolic rate, and 42 per cent reduced reactive oxygen species (ROS) production, compared with freshly ejaculated sperm. Rank differences between males in ROS production and metabolic rate observed in ejaculated sperm did not predict rank differences in stored sperm. Our method of simultaneously measuring ROS production and metabolic rate of the same sample has the advantage of providing data that are independent of sperm density and any extracellular antioxidants that are proteins. Our method also excludes effects owing to accumulated hydrogen peroxide. Our results unify aspects of competing theories of cellular ageing and suggest that reducing metabolic rate may be an important means of extending stored sperm lifespan and functionality in crickets. Our data also provide a possible explanation for why traits of ejaculates sampled from the male may be rather poor predictors of paternity in sexual selection studies and likelihood of pregnancy in reproductive medicine

Potentialisation de la chimiothérapie en milieu oxygéné (implication des radicaux libres dans l'effet des anthracyclines)

L hypoxie tumorale peut induire une résistance aux traitements par l adriamycine (ADR), et l effet anti-cancéreux de l anthracycline peut augmenter sous oxygénation hyperbare. Cependant, les mécanismes d action impliqués dans ce gain d efficacité thérapeutique restent à élucider.Nous avons évalué l implication de la production d espèces réactives de l oxygène (ROS) dans l amélioration de l effet de l ADR sur des cellules lymphoblastiques humaines CCRF-CEM sous hypoxie (2% O2) et sous normoxie (21% O2). Nous avons utilisé une nouvelle méthode de détection de ROS basée sur la mesure de la durée de vie de fluorescence de l'acide 1-pyrène butyrique. L analyse d images numériques des populations cellulaires après triple-marquage a fourni des informations morphométriques (tailles cellulaire et nucléaire) et fonctionnelles (activité mitochondriale, teneur en ADN) permettant (i) de quantifier l induction de l apoptose, et (ii) d établir la distribution du cycle cellulaire par analyse multiparamétrique des données.Nous avons observé que le blocage du cycle cellulaire par l'ADR ne dépend pas des conditions d'oxygénation, alors que l induction de l apoptose et la production de ROS dues au traitement sont plus importantes sous condition oxygénée (21% O2). Si on admet que la condition normoxique est une hyperoxygénation comparée à l état d hypoxie tumorale in vivo, alors le gain d efficacité thérapeutique de l ADR fournit par une oxygénation hyperbare pourrait résulter d'une plus forte production de ROS par l'anthracycline, qui entraînerait une induction des processus apoptotiques plus importante.Tumour hypoxia is causally related with resistance to adriamycin (ADR) treatment. However, how hyperbaric oxygen therapy leads to therapeutic gain of the drug is unclear.We investigated the relation of reactive oxygen species (ROS) generation with anti-tumoural effect of ADR on human lymphoblastic CCRF-CEM cells under hypoxic (2% O2) and normoxic (21% O2) conditions. A new method was used to measure intracellular ROS variations through the fluorescence lifetime of 1-pyrenebutyric acid. Numerical image analysis of cell populations labelled with different vital stains allowed to collect morphometric (cellular and nuclear sizes) and physiological (mitochondrial activity, DNA content) informations used to (i) quantify apoptosis induction, and (ii) determine the cell cycle distribution through multiparametric analysis of collected data.We observed that oxygen level has no effect on the cell cycle arrest induced by ADR, whereas apoptosis induction and ROS production resulting from treatment are higher under oxygenated conditions (i.e. normoxia). Considering normoxia as a hyperoxygenated condition compared to in vivo hypoxic tumour level, we suggested that improvement of anti-cancerous effect of ADR due to hyperbaric oxygen therapy results from higher intracellular ROS generation by the drug, leading to a greater induction of apoptosis.PERPIGNAN-BU Sciences (661362101) / SudocSudocFranceF

Quenching of long lifetime emitting fluorophores with paramagnetic molecules

International audienceIn this work, we have studied quenching of the fluorescence of two well-known oxygen probes, 1-pyrene butyric acid (PBA) and tris(2,2'-bipyridine)ruthenium ([Ru(bpy)(3)](2+)) by reactive oxygen species (superoxide anion, nitric oxide derivative, hydrogen peroxide) and by the O(2) molecule. Both, time-resolved and steady state fluorescence measurements were performed in solution (ethanol, dimethyl sufoxide, water) and in micelles of Sodium Dodecyl Sulfate that serve as a model for membrane-containing biological structures. We have found that only the free radicals and O(2) can actively quench for the two probes, but not the diamagnetic H(2)O(2). Our data correspond to the classical Stern-Volmer equation. H(2)O(2) has an effect only at high molar concentrations (>0.1 M). In contrast, effective concentrations of free radicals and O(2) that lead to quenching are in millimolar range. In conclusion, our methods allows for detecting global ROS that are small free radicals without interference from the reactive hydroxyl radical. Our data suggest that the method can be used for the quantification of ROS in individual living cells based on the measurement of fluorescence lifetime of those probe

A comparative study of Caesalpinia bonduc (L.) Roxb. root extracts on sexual behaviour in male Wistar rats

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Somatic production of reactive oxygen species does not predict its production in sperm cells across Drosophila melanogaster lines

International audienceAbstract Objective Sperm ageing has major evolutionary implications but has received comparatively little attention. Ageing in sperm and other cells is driven largely by oxidative damage from reactive oxygen species (ROS) generated by the mitochondria. Rates of organismal ageing differ across species and are theorized to be linked to somatic ROS levels. However, it is unknown whether sperm ageing rates are correlated with organismal ageing rates. Here, we investigate this question by comparing sperm ROS production in four lines of Drosophila melanogaster that have previously been shown to differ in somatic mitochondrial ROS production, including two commonly used wild-type lines and two lines with genetic modifications standardly used in ageing research. Results Somatic ROS production was previously shown to be lower in wild-type Oregon-R than in wild-type Dahomey flies; decreased by the expression of alternative oxidase (AOX), a protein that shortens the electron transport chain; and increased by a loss-of-function mutation in dj-1 β, a gene involved in ROS scavenging. Contrary to predictions, we found no differences among these four lines in the rate of sperm ROS production. We discuss the implications of our results, the limitations of our study, and possible directions for future research

Some pathophysiological insights into ovarian infestation by Myxobolus sp. (Myxozoa: Myxosporea) in Clarias gariepinus (Clariids: Silurids) from Bénin (West Africa)

International audienceMature female specimens of the catfish Clarias gariepinus originating from Ouémé River (Benin) were investigated into ovarian myxozoan parasites. Spores of Myxobolus sp. (Myxozoa: Myxosporea) were found encrusted in the whitish color oocytes which present fat dot aspect in the gonads. The pathological investigation by electron microscopy revealed that maturation and multiplication of spores induced lytic action, deformation and dysfunction of the oocyte internal structures. No host inflammatory reaction was observed, while yolk, lipid, mitochondria, and other oocyte components were degenerated inducing empty area in the oocyte and could lead to castration in case of wide infestation. The mean prevalence was 19.79 %. No significant difference was observed within seasonal prevalence (χ2 = 1.771; df = 3; p > 0.05). Though the host length classes ranging from 35 to 39 cm and 40 to 45 cm were more infected, difference was not significant (χ2 = 2.273; df = 4; p > 0.05) within them. The spores are ovoid in shape with two polar capsules which are equal in size, pyriform, and converging in anterior part of spore with four to five polar filament turns. Spore body are (11.47 ± 0.67) × (8.19 ± 0.52) μm length by width while polar capsule size are (4.24 ± 0.25) × (3.07 ± 0.28) μm and located in the first third portion of the spore. The molecular approaches are still running for accurate identification of this parasite

Oxidative stress does not play a primary role in the toxicity induced with clinical doses of doxorubicin in myocardial H9c2 cells

International audienceThe implication of oxidative stress as primary mechanism inducing doxorubicin (DOX) cardiotoxicity is still questionable as many in vitro studies implied supra-clinical drug doses or unreliable methodologies for reactive oxygen species (ROS) detection. The aim of this study was to clarify whether oxidative stress is involved in compliance with the conditions of clinical use of DOX, and using reliable tools for ROS detection. We examined the cytotoxic mechanisms of 2 μM DOX 1 day after the beginning of the treatment in differentiated H9c2 rat embryonic cardiac cells. Cells were exposed for 2 or 24 h with DOX to mimic a single chronic dosage or to favor accumulation, respectively. We found that apoptosis was prevalent in cells exposed for a short period with DOX: cells showed typical hallmarks as loss of anchorage ability, mitochondrial hyperpolarization followed by the collapse of mitochondrial activity, and nuclear condensation. Increasing the exposure period favored a shift to necrosis as the cells preferentially exhibited early DNA impairment and nuclear swelling. In either case, measuring the fluorescence lifetime of 1-pyrenebutyric acid or the intensities of dihydroethidium or amplex red showed a consistent pattern in ROS production which was a slight increased level far from representative of an oxidative stress. Moreover, pre-treatment with dexrazoxane provided a cytoprotective effect although it failed to detoxify ROS. Our data support that oxidative stress is unlikely to be the primary mechanism of DOX cardiac toxicity in vitro

Peptide Vectors Carry Pyrene to Cell Organelles Allowing Real‐Time Quantification of Free Radicals in Mitochondria by Time‐Resolved Fluorescence Microscopy

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