Patterns of MTT reduction in mammalian spermatozoa

Reproduction ◽  
2020 ◽  
Vol 160 (3) ◽  
pp. 431-445
Author(s):  
Robert John Aitken ◽  
Diatsendoula Gregoratos ◽  
Leslie Kutzera ◽  
Emma Towney ◽  
Minjie Lin ◽  
...  
Keyword(s):  
Cell Types ◽  
Sperm Head ◽  
Human Spermatozoa ◽  
Redox Activity ◽  
Mitochondrial Activity ◽  
Oxidoreductase Activity ◽  
Significant Information ◽  
Diphenylene Iodonium ◽  
Mtt Reduction ◽  
Mammalian Spermatozoa

MTT is widely used in biology as a probe for cell viability by virtue of its ability to generate deposits of insoluble formazan at sites of intense oxidoreductase activity. This response is generally held to reflect mitochondrial redox activity; however, extra-mitochondrial MTT reduction has also been recorded in certain cell types. Given this background, we set out to determine the major sites of formazan deposition in mammalian spermatozoa. In the mouse, most MTT reduction took place within the extensive mitochondrial gyres, with a single minor site of formazan deposition on the sperm head. By contrast, human spermatozoa generally displayed small disorganized midpieces exhibiting moderate MTT reduction activity accompanied by a major extra-mitochondrial formazan deposit on various locations in the sperm head from the neck to the anterior acrosome. Equine spermatozoa presented a combination of these two patterns, with major formazan deposition in the mitochondria accompanied by an extra-mitochondrial formazan deposit in around 20% of cells. The functionality of human spermatozoa was positively associated with the presence of an extra-mitochondrial formazan granule. Subsequent studies indicated that this extra-mitochondrial activity was suppressed by the presence of diphenylene iodonium, zinc, 2-deoxyglucose, co-enzyme Q, an SOD mimetic and NADPH oxidase inhibitors. We conclude that the pattern of MTT reduction to formazan by spermatozoa is species specific and conveys significant information about the relative importance of mitochondrial vs extra-mitochondrial redox activity that, in turn, defines the functional qualities of these cells.

The Fine Structure of Glycocalyx in Human Spermatozoa. A High Resolution Cytochemical Study

1973 ◽  
Vol 31 ◽  
pp. 640-641
Author(s):  
P. Hernández-Jáuregui ◽  
A. Sosa ◽  
A. González Angulo
Keyword(s):  
Negative Charge ◽  
Iron Hydroxide ◽  
Human Spermatozoa ◽  
Surface Coat ◽  
Cell Surfaces ◽  
Colloidal Iron ◽  
Cytochemical Study ◽  
Specific Permeability ◽  
Extracellular Glycoprotein ◽  
Mammalian Spermatozoa

Glycocalyx is the name given by Bennett to the extracellular glycoprotein coat present in some cell surfaces. It appears to play an important role in cell properties such as antigenicity, cell adhesivity, specific permeability, and ATP ase activity. In the sperm this coat can be directly related to such important phenomena as capacitation and fertilization. The presence of glycocalyx in invertebrate spermatozoa has already been demonstrated. Recently Yanagimachi et al. has determined the negative charges on sperm surfaces of mammalian spermatozoa including man, using colloidal iron hydroxide. No mention was made however of the outer surface coat as composed of substances other than those confering a negative charge. The purpose of this work was therefore to determine the presence of a glycocalyx in human spermatozoa using alcian blue and lanthanum staining.

scholarly journals Enzymatic Degradation of Phenazines Can Generate Energy and Protect Sensitive Organisms from Toxicity

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Kyle C. Costa ◽  
Megan Bergkessel ◽  
Scott Saunders ◽  
Jonas Korlach ◽  
Dianne K. Newman
Keyword(s):  
Microbial Communities ◽  
Pathogenic Fungi ◽  
Cell Types ◽  
Redox Activity ◽  
Mycobacterium Fortuitum ◽  
Active Metabolites ◽  
Content Type ◽  
Phenazine Production ◽  
Physiological Benefits

ABSTRACTDiverse bacteria, including severalPseudomonasspecies, produce a class of redox-active metabolites called phenazines that impact different cell types in nature and disease. Phenazines can affect microbial communities in both positive and negative ways, where their presence is correlated with decreased species richness and diversity. However, little is known about how the concentration of phenazines is modulatedin situand what this may mean for the fitness of members of the community. Through culturing of phenazine-degrading mycobacteria, genome sequencing, comparative genomics, and molecular analysis, we identified several conserved genes that are important for the degradation of threePseudomonas-derived phenazines: phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), and pyocyanin (PYO). PCA can be used as the sole carbon source for growth by these organisms. Deletion of several genes inMycobacterium fortuitumabolishes the degradation phenotype, and expression of two genes in a heterologous host confers the ability to degrade PCN and PYO. In cocultures with phenazine producers, phenazine degraders alter the abundance of different phenazine types. Not only does degradation support mycobacterial catabolism, but also it provides protection to bacteria that would otherwise be inhibited by the toxicity of PYO. Collectively, these results serve as a reminder that microbial metabolites can be actively modified and degraded and that these turnover processes must be considered when the fate and impact of such compounds in any environment are being assessed.IMPORTANCEPhenazine production byPseudomonasspp. can shape microbial communities in a variety of environments ranging from the cystic fibrosis lung to the rhizosphere of dryland crops. For example, in the rhizosphere, phenazines can protect plants from infection by pathogenic fungi. The redox activity of phenazines underpins their antibiotic activity, as well as providing pseudomonads with important physiological benefits. Our discovery that soil mycobacteria can catabolize phenazines and thereby protect other organisms against phenazine toxicity suggests that phenazine degradation may influence turnoverin situ. The identification of genes involved in the degradation of phenazines opens the door to monitoring turnover in diverse environments, an essential process to consider when one is attempting to understand or control communities influenced by phenazines.

scholarly journals Mitochondrial Retrograde Signaling Contributes to Metabolic Differentiation in Yeast Colonies

2021 ◽  
Vol 22 (11) ◽  
pp. 5597
Author(s):  
Vítězslav Plocek ◽  
Kristýna Fadrhonc ◽  
Jana Maršíková ◽  
Libuše Váchová ◽  
Alexandra Pokorná ◽  
...  
Keyword(s):  
Cell Types ◽  
Colony Development ◽  
Mitochondrial Activity ◽  
Mitochondrial Translation ◽  
Metabolic Intermediates ◽  
Nutritional Conditions ◽  
Mitochondrial Ribosome ◽  
Development And Differentiation ◽  
Cell Subpopulations ◽  
Different Cell Types

During development of yeast colonies, various cell subpopulations form, which differ in their properties and specifically localize within the structure. Three branches of mitochondrial retrograde (RTG) signaling play a role in colony development and differentiation, each of them activating the production of specific markers in different cell types. Here, aiming to identify proteins and processes controlled by the RTG pathway, we analyzed proteomes of individual cell subpopulations from colonies of strains, mutated in genes of the RTG pathway. Resulting data, along with microscopic analyses revealed that the RTG pathway predominantly regulates processes in U cells, long-lived cells with unique properties, which are localized in upper colony regions. Rtg proteins therein activate processes leading to amino acid biosynthesis, including transport of metabolic intermediates between compartments, but also repress expression of mitochondrial ribosome components, thus possibly contributing to reduced mitochondrial translation in U cells. The results reveal the RTG pathway’s role in activating metabolic processes, important in U cell adaptation to altered nutritional conditions. They also point to the important role of Rtg regulators in repressing mitochondrial activity in U cells.

scholarly journals NADPH-dependent extracellular superoxide production is vital to photophysiology in the marine diatom Thalassiosira oceanica

2019 ◽  
Vol 116 (33) ◽  
pp. 16448-16453 ◽  
Author(s):  
Julia M. Diaz ◽  
Sydney Plummer ◽  
Colleen M. Hansel ◽  
Peter F. Andeer ◽  
Mak A. Saito ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Cell Types ◽  
Redox Balance ◽  
Superoxide Production ◽  
Marine Phytoplankton ◽  
Marine Diatom ◽  
High Sequence Similarity ◽  
Whole Cells ◽  
Wide Range ◽  
Diphenylene Iodonium

Reactive oxygen species (ROS) like superoxide drive rapid transformations of carbon and metals in aquatic systems and play dynamic roles in biological health, signaling, and defense across a diversity of cell types. In phytoplankton, however, the ecophysiological role(s) of extracellular superoxide production has remained elusive. Here, the mechanism and function of extracellular superoxide production by the marine diatom Thalassiosira oceanica are described. Extracellular superoxide production in T. oceanica exudates was coupled to the oxidation of NADPH. A putative NADPH-oxidizing flavoenzyme with predicted transmembrane domains and high sequence similarity to glutathione reductase (GR) was implicated in this process. GR was also linked to extracellular superoxide production by whole cells via quenching by the flavoenzyme inhibitor diphenylene iodonium (DPI) and oxidized glutathione, the preferred electron acceptor of GR. Extracellular superoxide production followed a typical photosynthesis-irradiance curve and increased by 30% above the saturation irradiance of photosynthesis, while DPI significantly impaired the efficiency of photosystem II under a wide range of light levels. Together, these results suggest that extracellular superoxide production is a byproduct of a transplasma membrane electron transport system that serves to balance the cellular redox state through the recycling of photosynthetic NADPH. This photoprotective function may be widespread, consistent with the presence of putative homologs to T. oceanica GR in other representative marine phytoplankton and ocean metagenomes. Given predicted climate-driven shifts in global surface ocean light regimes and phytoplankton community-level photoacclimation, these results provide implications for future ocean redox balance, ecological functioning, and coupled biogeochemical transformations of carbon and metals.

scholarly journals Mitochondrial Target of Nobiletin's Action

2016 ◽  
Vol 11 (12) ◽  
pp. 1934578X1601101 ◽  
Author(s):  
Nino Sharikadze ◽  
Natia Jojua ◽  
Maia Sepashvili ◽  
Elene Zhuravliova ◽  
David G Mikeladze
Keyword(s):  
Oxygen Consumption ◽  
Membrane Potential ◽  
Cell Types ◽  
Bovine Brain ◽  
Oxidoreductase Activity ◽  
Substrate Level Phosphorylation ◽  
Mitochondrial Target ◽  
Substrate Level ◽  
Transient Elevation ◽  
Ketoglutarate Dehydrogenase

Nobiletin is an O-methylated flavonoid found in citrus peels that have anticancer, antiviral, neuroprotective, anti-inflammatory activities and depending on the cell types exhibits both pro- or anti-apoptotic properties. We have found that nobiletin decreases oxygen consumption by bovine brain isolated mitochondria in the presence of glutamate and malate and increases in the presence of succinate. In parallel, nobiletin increases NADH oxidation, a-ketoglutarate dehydrogenase activities and through matrix substrate-level phosphorylation elevates the a-ketoglutarate-dependent production of ATP. In addition, nobiletin reduces the production of peroxides in the presence of complex I substrates and slightly enhances succinate-driven H2O2 formation. Besides, nobiletin induces transient elevation of membrane potential followed by mild depolarization. Affinity purified nobiletin binding proteins revealed one major anti-NDUFV1 positive protein with 52kD and NADH: ubiquinone oxidoreductase activity. This fraction can produce peroxide that is inhibited by nobiletin. We propose that nobiletin may act as a mild “uncoupler”, which through activation of a-ketoglutarate dehydrogenase (a-KGDH)-complex and acceleration of matrix substrate-level phosphorylation maintains membrane potential at an abnormal level. This switch in mitochondrial metabolism could elevate succinate-driven oxygen consumption that may underlay in both pro- and anti-apoptotic effects of nobiletin.

scholarly journals Species difference in the expression of Fas and Fas ligand in mature mammalian spermatozoa

2017 ◽  
Vol 62 (No. 12) ◽  
pp. 519-526
Author(s):  
Y. Li ◽  
M. Sun ◽  
J. Zhu ◽  
G. Jiao ◽  
J. Lin ◽  
...  
Keyword(s):  
Fas Ligand ◽  
Species Difference ◽  
Female Genital Tract ◽  
Human Spermatozoa ◽  
Whole Body ◽  
Fasl Expression ◽  
Weak Signals ◽  
Boar Spermatozoa ◽  
Mature Spermatozoa ◽  
Mammalian Spermatozoa

Although it has been proposed that the Fas and Fas ligand (FasL) may protect ejaculated spermatozoa against apoptosis induced by lipoperoxidative damage and against lymphocytes present in the female genital tract, studies reported conflicting results on the presence of Fas receptors in ejaculated human spermatozoa. Furthermore, the expression of Fas/FasL on mature spermatozoa has not been observed in several important mammals. Using seven species, we observed the possibility for species difference in Fas/FasL expression on mature spermatozoa by both immunofluorescence microscopy and western blot analysis. Whereas intensive signals of Fas immunolabelling were detected in sperm head and middle piece and weak signals observed in the tail in 86–100% of the mouse, rat, bull, ram, and buck spermatozoa, only weak signals were detected on the whole body of 27% boar spermatozoa and in the head of 21% human spermatozoa. The pattern of FasL localization was identical to that of Fas in spermatozoa from human, mouse, rat, ram, and buck, but boar and bull spermatozoa showed weak and intensive FasL signals, respectively, only in the head. Western blotting further confirmed the Fas and FasL expression in mouse, rat, bull, ram, and buck, but not in human and boar spermatozoa. Taken together, the results revealed a marked species difference in Fas/FasL expression and an extensive co-expression of Fas and FasL among mature mammalian spermatozoa, suggesting that whereas spermatozoa from most species may be protected by Fas/FasL, those from human and boar may not use the Fas system for protection.

Unprotected freezing of human spermatozoa exerts a detrimental effect on their oocyte activating capacity and chromosome integrity

Zygote ◽  
1996 ◽  
Vol 4 (04) ◽  
pp. 263-268 ◽  
Author(s):  
Andrei V. Rybouchkin ◽  
Paul De Sutter ◽  
Marc Dhont
Keyword(s):  
Assisted Reproduction ◽  
Detrimental Effect ◽  
Chromosomal Abnormalities ◽  
Ethanol Solution ◽  
Human Spermatozoa ◽  
Physical Factors ◽  
Reproductive Characteristics ◽  
Chromosome Integrity ◽  
The Stability ◽  
Mammalian Spermatozoa

SummaryThe influence of unprotected freezing of mammalian spermatozoa on their oocyte activating capacity and chromosome integrity is unknown. However, this type of sperm treatment has been used in assisted reproduction by intracytoplasmic sperm injection in cattle and humans. The mouse oocyte injection test was used to analyse the influence of unprotected freezing of human spermatozoa on their reproductive characteristics. Mouse oocytes were microinjected with intact human spermatozoa or spermatozoa treated with two cycles of unprotected freeze-thawing. Oocytes surviving the injection were either cultured without further treatment or exposed to ethanol solution to induce parthenogenetic activation. Both injected and activated oocytes were used for sperm chromosome analysis. The results revealed a significant reduction in oocyte activating capacity and a tenfold increase in the incidence of structural chromosomal abnormalities in human spermatozoa treated by unprotected freezing. We conclude that unprotected freezing of human spermatozoa has a detrimental effect on their reproductive characteristics. Our data also provide a new perspective on the stability of mammalian spermatozoa to physical factors and demonstrate the importance of detailed analysis of the stability of sperm structures for successful development of new approaches in assisted reproduction.

scholarly journals Plasma membrane electron transport in pancreatic β-cells is mediated in part by NQO1

2011 ◽  
Vol 301 (1) ◽  
pp. E113-E121 ◽  
Author(s):  
Joshua P. Gray ◽  
Timothy Eisen ◽  
Gary W. Cline ◽  
Peter J. S. Smith ◽  
Emma Heart
Keyword(s):  
Plasma Membrane ◽  
Electron Transport ◽  
Potassium Cyanide ◽  
Cell Types ◽  
Glycolytic Flux ◽  
Dependent Manner ◽  
Β Cells ◽  
Quinone Oxidoreductase ◽  
Plasma Membrane Electron Transport ◽  
Diphenylene Iodonium

Plasma membrane electron transport (PMET), a cytosolic/plasma membrane analog of mitochondrial electron transport, is a ubiquitous system of cytosolic and plasma membrane oxidoreductases that oxidizes cytosolic NADH and NADPH and passes electrons to extracellular targets. While PMET has been shown to play an important role in a variety of cell types, no studies exist to evaluate its function in insulin-secreting cells. Here we demonstrate the presence of robust PMET activity in primary islets and clonal β-cells, as assessed by the reduction of the plasma membrane-impermeable dyes WST-1 and ferricyanide. Because the degree of metabolic function of β-cells (reflected by the level of insulin output) increases in a glucose-dependent manner between 4 and 10 mM glucose, PMET was evaluated under these conditions. PMET activity was present at 4 mM glucose and was further stimulated at 10 mM glucose. PMET activity at 10 mM glucose was inhibited by the application of the flavoprotein inhibitor diphenylene iodonium and various antioxidants. Overexpression of cytosolic NAD(P)H-quinone oxidoreductase (NQO1) increased PMET activity in the presence of 10 mM glucose while inhibition of NQO1 by its inhibitor dicoumarol abolished this activity. Mitochondrial inhibitors rotenone, antimycin A, and potassium cyanide elevated PMET activity. Regardless of glucose levels, PMET activity was greatly enhanced by the application of aminooxyacetate, an inhibitor of the malate-aspartate shuttle. We propose a model for the role of PMET as a regulator of glycolytic flux and an important component of the metabolic machinery in β-cells.

Free radicals, lipid peroxidation and sperm function

1995 ◽  
Vol 7 (4) ◽  
pp. 659 ◽  
Author(s):  
RJ Aitken
Keyword(s):  
Reactive Oxygen Species ◽  
Lipid Peroxidation ◽  
Reactive Oxygen ◽  
Human Spermatozoa ◽  
Sperm Function ◽  
Oxygen Species ◽  
Normal Sperm ◽  
Oxidative Processes ◽  
Mammalian Spermatozoa

The cellular generation of reactive oxygen species was first observed in mammalian spermatozoa in the late 1940s. The field then remained dormant for 30 years until Thaddeus Mann and Roy Jones published a series of landmark papers in the 1970s in which the importance of lipid peroxidation as a mechanism for damaging mammalian spermatozoa was first intimated. The subsequent demonstration that human spermatozoa produce reactive oxygen species and are susceptible to peroxidative damage has triggered intense interest in the role of oxidative stress in the aetiology of male infertility. Moreover, data have recently been obtained to indicate that, although excessive exposure to reactive oxygen species may be harmful to spermatozoa, in physiological amounts these molecules are of importance in the control of normal sperm function. This review considers the dualistic role of reactive oxygen species and sets out the current understanding of the importance of oxidative processes in both the physiology and the pathology of the human spermatozoon. Extra keywords: human spermatozoa, reactive oxygen species.

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