Development of the NBT assay as a marker of sperm oxidative stress

Tunc, Ozlem; Thompson, Jeremy G.; Tremellen, Kelton

doi:10.1111/j.1365-2605.2008.00941.x

Cited by 83 publications

(64 citation statements)

References 33 publications

(83 reference statements)

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“…NAC treatment also suppressed mineralization of the differentiated MC3T3-E1 cells in a dose-dependent manner (Fig. 3B), suggesting that ROS play an important quantitative NBT (nitro blue tetrazolium) test was used to measure ROS production within an intracellular compartment as described (11,26). MC3T3-E1 cells were plated in 12-well plates and incubated with or without NAC.…”

Section: Involvement Of Ros In Osteoblastic Differentiation Of Mc3t3-mentioning

confidence: 92%

Involvement of reactive oxygen species in osteoblastic differentiation of MC3T3-E1 cells accompanied by mitochondrial morphological dynamics

et al. 2013

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Bone remodeling is regulated by local factors that regulate bone-forming osteoblasts and boneresorbing osteoclasts, in addition to hormonal activity. Recent studies have shown that reactive oxygen species (ROS) act as an intracellular signal mediator for osteoclast differentiation. However the role of ROS on osteoblast differentiation is poorly understood. Here, we investigated the impact of ROS on osteoblastic differentiation of MC3T3-E1 cells. Osteogenic induction resulted in notable enhancement of mineralization and expression of osteogenic marker gene alkaline phosphatase, which were accompanied by an increase in ROS production. Additionally, we found that mitochondrial morphology dynamically changed from tubular reticulum to fragmented structures during the differentiation, suggesting that mitochondrial morphological transition is a novel osteoblast differentiation index. The antioxidant N-acetyl cysteine prevented not only ROS production but also mineralization and mitochondrial fragmentation. It is therefore suggested that the ROSdependent signaling pathways play a role in osteoblast differentiation accompanied by mitochondrial morphological transition.Bone is a dynamic organ that undergoes continuous remodeling while maintaining a balance between bone formation and resorbtion. Osteoblasts, which synthesize and mineralize new bone, and osteoclasts, which resorb bone, act in concert to maintain bone homeostasis. Bone mass density is maintained constant under the control of multiple systemic and local factors such as sex hormones, parathyroid hormone, growth hormone, and proinflammatory cytokines (21,22,25). Imbalanced functions of these two activities are involved in various types of bone diseases such as osteoporosis and vascular calcification, which are the major age-related diseases. To understand the pathogenic mechanism of these diseases, it is important to elucidate the regulatory mechanisms of the differentiation and activation of osteoclasts and osteoblasts. Although the physiological mechanisms of bone metabolism are becoming better understood, the intracellular signal transduction pathway that contributes to the regulation of differentiation and activation of osteoclasts and osteoblasts remains to be established. In this regard, it was recently reported that reactive oxygen species (ROS) act as an intracellular signal mediator for osteoclast differentiation, in which RANKL (receptor activator of NF-κB (nuclear factor κB) ligand) induces NADPH oxidase-derived ROS as an essential mechanism for osteoclast differentiation (10,15,18). However, the role of ROS on osteoblast differentiation remains largely uncertain. Mitochondria are crucial organelles involved in cellular energy production and in the regulation of numerous aspects of cellular activity including Ca 2+ signaling and apoptosis (5, 23). Furthermore, it has become evident in recent years that mitochondria

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Section: Involvement Of Ros In Osteoblastic Differentiation Of Mc3t3-mentioning

confidence: 92%

Involvement of reactive oxygen species in osteoblastic differentiation of MC3T3-E1 cells accompanied by mitochondrial morphological dynamics

et al. 2013

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“…The NBT assay for oxidative stress was adapted from Tunc et al (2009). For the assay, 5 9 10 6 cells in 200 lL BWW were incubated at 37°C with ROS-generating reagents for the periods of time stipulated above (15 min for all treatments other than 4HNE for which a 1 h incubation period was used).…”

Section: Nitroblue Tetrazolium Assaymentioning

confidence: 99%

On methods for the detection of reactive oxygen species generation by human spermatozoa: analysis of the cellular responses to catechol oestrogen, lipid aldehyde, menadione and arachidonic acid

et al. 2013

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SummaryOxidative stress is known to have a major impact on human sperm function and, as a result, there is a need to develop sensitive methods for measuring reactive oxygen species (ROS) generation by these cells. A variety of techniques have been developed for this purpose including chemiluminescence (luminol and lucigenin), flow cytometry (MitoSOX Red, dihydroethidium, 4,5‐diaminofluorescein diacetate and 2′,7′‐dichlorodihydrofluorescein diacetate) and spectrophotometry (nitroblue tetrazolium). The relative sensitivity of these assays and their comparative ability to detect ROS generated in different subcellular compartments of human spermatozoa, have not previously been investigated. To address this issue, we have compared the performance of these assays when ROS generation was triggered with a variety of reagents including 2‐hydroxyestradiol, menadione, 4‐hydroxynonenal and arachidonic acid. The results revealed that menadione predominantly induced release of ROS into the extracellular space where these metabolites could be readily detected by luminol‐peroxidase and, to a lesser extent, 2′,7′‐dichlorodihydrofluorescein. However, such sensitivity to extracellular ROS meant that these assays were particularly vulnerable to interference by leucocytes. The remaining reagents predominantly elicited ROS generation by the sperm mitochondria and could be optimally detected by MitoSOX Red and DHE. Examination of spontaneous ROS generation by defective human spermatozoa revealed that MitoSOX Red was the most effective indicator of oxidative stress, thereby emphasizing the general importance of mitochondrial dysregulation in the aetiology of defective sperm function.

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“…In human sperm motility, morphology and concentration are standard parameters for assessing male fertility (86). Levels of sperm DNA damage and ROS have more recently been used as a marker of sperm health, with increased levels in humans correlating with reduced fertilization, embryonic development, and pregnancy loss (4,11,17,27,80,88). This suggests that diet and/or exercise could significantly improve fertilization, blastocyst development, and pregnancy rates in obese males by improving sperm motility, morphology, levels of sperm DNA damage, and sperm mitochondrial ROS.…”

Section: E775 Diet and Exercise Improve Sperm Function In Obese Micementioning

confidence: 99%

Diet and exercise in an obese mouse fed a high-fat diet improve metabolic health and reverse perturbed sperm function

Palmer¹,

Bakos

Owens³

et al. 2012

American Journal of Physiology-Endocrinology and Metabolism

198

181

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Palmer NO, Bakos HW, Owens JA, Setchell BP, Lane M. Diet and exercise in an obese mouse fed a high-fat diet improve metabolic health and reverse perturbed sperm function. Am J Physiol Endocrinol Metab 302: E768 -E780, 2012. First published January 17, 2012; doi:10.1152/ajpendo.00401.2011.-Male obesity is associated with reduced sperm motility and morphology and increased sperm DNA damage and oxidative stress; however, the reversibility of these phenotypes has never been studied. Therefore, the aim of this study was to assess the reversibility of obesity and its associated sperm physiology and function in mice in response to weight loss through diet and exercise. C57BL6 male mice (n ϭ 40) were fed either a control diet (CD; 6% fat) or a high-fat diet (HFD; 21% fat) for 10 wk before allocation to either diet and/or swimming exercise interventions for 8 wk. Diet alone reduced adiposity (1.6-fold) and serum cholesterol levels (1.7-fold, P Ͻ 0.05), while exercise alone did not alter these, but exercise plus diet also improved glucose tolerance (1.3-fold, P Ͻ 0.05). Diet and/or exercise improved sperm motility (1.2-fold) and morphology (1.1-fold, P Ͻ 0.05), and reduced sperm DNA damage (1.5-fold), reactive oxygen species (1.1-fold), and mitochondrial membrane potential (1.2-fold, P Ͻ 0.05) and increased sperm binding (1.4-fold) (P Ͻ 0.05). Sperm parameters were highly correlated with measures of glycemia, insulin action, and serum cholesterol (all P Ͻ 0.05) regardless of adiposity or intervention, suggesting a link between systemic metabolic status and sperm function. This is the first study to show that the abnormal sperm physiology resulting from obesity can be reversed through diet and exercise, even in the presence of ongoing obesity, suggesting that diet and lifestyle interventions could be a combined approach to target subfertility in overweight and obese men. lifestyle interventions; fertility and metabolism OBESITY IS A GLOBAL HEALTH PROBLEM that is reaching epidemic proportions, with 1.6 billion adults classified as overweight and 400 million adults classified as obese worldwide (60). Consequently, the rates of male obesity in reproductive age men has nearly tripled since the 1970s (33). Recent studies in both humans and other species shows that male obesity impairs male reproductive potential, affecting both the molecular and physical structure of sperm (1,10,16,20,21,34,35,38,45,47,50,63,75,79) and the health of the developing fetus and subsequent offspring (9,24,42,55,59).Although fertility is not always impaired in obese men, almost 80% of men that present to fertility clinics are classified as either overweight or obese, suggestive of an interaction between obesity and sperm function (9). There is a negative impact of male obesity on standard semen parameters, including motility, count, and hormone abundance, such as testosterone and inhibin B (1,5,16,18,21,22,32,35,37,45,47,52,62,69,85). Further studies have reported reduced pregnancy success and live birth outcomes after IVF treatment in overweight and...

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Development of the NBT assay as a marker of sperm oxidative stress

Cited by 83 publications

References 33 publications

Involvement of reactive oxygen species in osteoblastic differentiation of MC3T3-E1 cells accompanied by mitochondrial morphological dynamics

Involvement of reactive oxygen species in osteoblastic differentiation of MC3T3-E1 cells accompanied by mitochondrial morphological dynamics

On methods for the detection of reactive oxygen species generation by human spermatozoa: analysis of the cellular responses to catechol oestrogen, lipid aldehyde, menadione and arachidonic acid

Diet and exercise in an obese mouse fed a high-fat diet improve metabolic health and reverse perturbed sperm function

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