TUNEL assay—Standardized method for testing sperm DNA fragmentation

Sharma, Rakesh; Iovine, Concetta; Agarwal, Ashok; Henkel, Ralf

doi:10.1111/and.13738

Cited by 46 publications

(41 citation statements)

References 101 publications

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“…Finally, 0.5 mL of propidium iodide (PI)/RNase solution was added, the specimens were transferred to a black 96-well plate and assessed with the Glomax Multi + combined spectro-fluoro-luminometer. TUNEL-positivity is expressed in % [ 69 ].…”

Section: Methodsmentioning

confidence: 99%

Possible Implications of Bacteriospermia on the Sperm Quality, Oxidative Characteristics, and Seminal Cytokine Network in Normozoospermic Men

Tvrdá

Lovíšek

Gálová

et al. 2022

IJMS

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This study focused on the identification of bacterial profiles of semen in normozoospermic men and their possible involvement in changes to the sperm structural integrity and functional activity. Furthermore, we studied possible fluctuations of selected cytokines, oxidative markers, and antibacterial proteins as a result of bacterial presence in the ejaculate. Sperm motility was assessed with computer-assisted sperm analysis, while sperm apoptosis, necrosis and acrosome integrity were examined with fluorescent methods. Reactive oxygen species (ROS) generation was quantified via luminometry, sperm DNA fragmentation was evaluated using the TUNEL protocol and chromatin-dispersion test, while the JC-1 assay was applied to evaluate the mitochondrial membrane potential. Cytokine levels were quantified with the biochip assay, whilst selected antibacterial proteins were quantified using the ELISA method. The predominant species identified by the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were Staphylococcus hominis, Staphylococcus capitis and Micrococcus luteus. The results revealed that the sperm quality decreased proportionally to the increasing bacterial load and occurrence of conditionally pathogenic bacteria, including Enterococcus faecalis, Staphylococcus aureus and Escherichia coli. Antimicrobial susceptibility tests revealed a substantial resistance of randomly selected bacterial strains to ampicillin, vancomycin, tobramycin, and tetracycline. Furthermore, an increased bacterial quantity in semen was accompanied by elevated levels of pro-inflammatory cytokines, including interleukin-1, interleukin-2, interleukin-6, tumor necrosis factor alpha as well as ROS overproduction and lipid peroxidation of the sperm membranes. Our results suggest that semen quality may be notably affected by the bacterial quantity as well as quality. It seems that bacteriospermia may be associated with inflammatory processes, oxidative stress, sperm structural deterioration, and a subsequent risk for the development of subfertility, even in normozoospermic males.

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Section: Methodsmentioning

confidence: 99%

Possible Implications of Bacteriospermia on the Sperm Quality, Oxidative Characteristics, and Seminal Cytokine Network in Normozoospermic Men

Tvrdá

Lovíšek

Gálová

et al. 2022

IJMS

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show abstract

“…Next, the tissue slides were stained with hematoxylin and eosin (H&E) in accordance with the standard procedure ( Liu et al, 2018 ). Furthermore, the tissue slides were analyzed using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assays ( Sharma et al, 2021 ) to evaluate the antitumor efficacy. Furthermore, blood samples of nude mice with different treatment were collected for biochemical analyses on the 21st day.…”

Section: Methodsmentioning

confidence: 99%

pH/Redox/Lysozyme-Sensitive Hybrid Nanocarriers With Transformable Size for Multistage Drug Delivery

Liu

Zhou

Liao

2022

Front. Bioeng. Biotechnol.

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The majority of current nanocarriers in cancer treatment fail to deliver encapsulated cargos to their final targets at therapeutic levels, which decreases the ultimate efficacy. In this work, a novel core–shell nanocarrier with a biodegradable property was synthesized for efficient drug release and subcellular organelle delivery. Initially, silver nanoparticles (AgNPs) were grafted with terminal double bonds originating from N, N′-bisacrylamide cystamine (BAC). Then, the outer coatings consisting of chitosan (CTS) and polyvinyl alcohol (PVA) were deposited on the surface of modified AgNPs using an emulsion method. To improve the stability, disulfide-containing BAC was simultaneously reintroduced to cross-link CTS. The as-prepared nanoparticles (CAB) possessed the desired colloidal stability and exhibited a high drug loading efficiency of cationic anticancer agent doxorubicin (DOX). Furthermore, CAB was tailored to transform their size into ultrasmall nanovehicles responding to weak acidity, high glutathione (GSH) levels, and overexpressed enzymes. The process of transformation was accompanied by sufficient DOX release from CAB. Due to the triple sensitivity, CAB enabled DOX to accumulate in the nucleus, leading to a great effect against malignant cells. In vivo assays demonstrated CAB loading DOX held excellent biosafety and superior antitumor capacity. Incorporating all the benefits, this proposed nanoplatform may provide valuable strategies for efficient drug delivery.

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“…The nucleus was stained with DAPI (2 μg/ml) away from light for 10 min (Shi et al, 2021). The Tunel staining images were photographed with a fluorescence microscope (Bio-tek, Winusky, VT) (Zhang et al, 2016;Li T. et al, 2019;Luo et al, 2019;Sharma et al, 2021).…”

Section: Tunel Analysismentioning

confidence: 99%

Validated Impacts of N6-Methyladenosine Methylated mRNAs on Apoptosis and Angiogenesis in Myocardial Infarction Based on MeRIP-Seq Analysis

Zhang

Hua

Dang

et al. 2022

Front. Mol. Biosci.

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Objectives: N6-methyladenosine (m6A) is hypothesized to play a role in the regulation of pathogenesis of myocardial infarction (MI). This study was designed to compare m6A-tagged transcript profiles to identify mRNA-specific changes on pathophysiological variations after MI.Methods: N6-methyladenosine methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were interacted to select m6A-modified mRNAs with samples collected from sham operated and MI rat models. m6A methylation regulated mRNAs were interacted with apoptosis/angiogenesis related genes in GeneCards. Afterwards, MeRIP-quantitative real-time PCR (MeRIP-qRT-PCR) was performed to measure m6A methylation level of hub mRNAs. m6A methylation variation was tested under different oxygen concentration or hypoxic duration in H9c2 cells and HUVECs. In addition, Western blot and qRT-PCR were employed to detect expression of hub mRNAs and relevant protein level. Flow cytometry and Tunel assay were conducted to assess apoptotic level. CCK-8, EdU, and tube formation assay were performed to measure cell proliferation and tube formation ability.Results: Upregulation of Mettl3 was firstly observed in vivo and in vitro, followed by upregulation of m6A methylation level. A total of 567 significantly changed m6A methylation peaks were identified, including 276 upregulated and 291 downregulated peaks. A total of 576 mRNAs were upregulated and 78 were downregulated. According to combined analysis of MeRIP-seq and RNA-seq, we identified 26 significantly hypermethylated and downregulated mRNAs. Based on qRT-PCR and interactive analysis, Hadh, Kcnn1, and Tet1 were preliminarily identified as hub mRNAs associated with apoptosis/angiogenesis. MeRIP-qRT-PCR assay confirmed the results from MeRIP-seq. With the inhibition of Mettl3 in H9c2 cells and HUVECs, downregulated m6A methylation level of total RNA and upregulated expression of hub mRNAs were observed. Increased m6A level was verified in the gradient context in terms of prolonged hypoxic duration and decreased oxygen concentration. Under simulated hypoxia, roles of Kcnn1 and Tet1 in angiogenesis and Hadh, Tet1, and Kcnn1 in apoptosis were further confirmed with our validation experiments.Conclusion: Roles of m6A-modified mRNA transcripts in the context of MI were preliminarily verified. In the context of m6A methylation, three hub mRNAs were validated to impact the process of apoptosis/angiogenesis. Our study provided theoretical basis and innovative targets for treatment of MI and paved the way for future investigations aiming at exploring upstream epigenetic mechanisms of pathogenesis after MI.

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TUNEL assay—Standardized method for testing sperm DNA fragmentation

Cited by 46 publications

References 101 publications

Possible Implications of Bacteriospermia on the Sperm Quality, Oxidative Characteristics, and Seminal Cytokine Network in Normozoospermic Men

Possible Implications of Bacteriospermia on the Sperm Quality, Oxidative Characteristics, and Seminal Cytokine Network in Normozoospermic Men

pH/Redox/Lysozyme-Sensitive Hybrid Nanocarriers With Transformable Size for Multistage Drug Delivery

Validated Impacts of N6-Methyladenosine Methylated mRNAs on Apoptosis and Angiogenesis in Myocardial Infarction Based on MeRIP-Seq Analysis

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