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•A service unit was established to remotely perform Sperm DNA Fragmentation (SDF) assessment (TUNEL) in a 2-step process.•Using this setting, a cut-off value of 9.17 % was determined ...in a fertile donor cohort.•Within a patient cohort, SDF negatively correlated with sperm number, vitality and motility, and positively with abnormal morphology and male age (P < 0.05).•TUNEL-positive cases depicted lower sperm quality and higher male age (P < 0.05).•A similar abnormal SDF incidence was determined among patients with or without semen abnormalities.
Infertility is a disease of the male or female reproductive systems. Male reproductive workup is based on routine semen analysis, although of limited value. The 2021 WHO Manual incorporated Sperm DNA Fragmentation (SDF) assessment, and highlighted the need for individual laboratories to define suitable thresholds. This study aimed to present an alternative to address this issue, determine an SDF cut-off value with fertile donors, and characterize SDF in a patient cohort and their relationship with semen parameters.
A service unit was established to remotely perform TUNEL assay in a 2 step-process. Semen samples were received at andrology laboratories, subjected to routine semen analysis (WHO, 2010), partially processed and transported to the service unit for SDF evaluation. Using this setting, studies were done in fertile donors (n = 15) to define the cut-off value, and in men undergoing infertility workup (n = 318).
A cut-off value of 9.17 % was determined with the fertile donor cohort. With this cut-off, a 64.46 % abnormal SDF incidence was determined in the patient cohort. SDF negatively correlated with sperm number, vitality and motility, and positively with abnormal morphology and male age (P < 0.05). TUNEL-positive cases depicted lower sperm quality and higher male age (P < 0.05). A similar abnormal SDF incidence was determined among patients with semen abnormalities. Asthenozoospermic and ≥40 years patient samples depicted higher (P < 0.05) SDF than those of the general population. SDF incidence was also high in normozoospermic patients.
Using a 2-step remote approach with a standardized procedure and an SDF cut-off value established with fertile donors, high SDF incidence in semen samples depicting normal and abnormal quality were identified in men consulting for infertility, highlighting the relevance of its evaluation as part of the male fertility workup.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Glioblastoma Multiforme (GBM), which is a common and primary brain tumor in adults, is an important cause of death worldwide as an aggressive and treatment-resistant cancer tumor. In this cell ...culture study, the apoptotic and anti-proliferative effects of borax and irinotecan at different doses, alone or in combination, were investigated in the YKG1 cell line. Cytotoxic activities were analyzed by MTT method and TUNEL staining after 24th and 48th hours of incubation with borax administered at doses of 1mg and 3mg per ml; irinotecan 50mM and 100mM. Both irinotecan and borax have been shown to induce apoptosis when used alone, and thus cause anti-proliferation. It was determined that these effects were potentiated by the combined application of the agents. In addition, it was determined that this effect in combined applications was more pronounced after 48 hours and at higher doses. In light of the data obtained, the combination of irinotecan with borax to increase the cytotoxic effect of irinotecan, which is used in many different cancer types, can be tried in further prospective studies.
Bisphenol A (BPA) is an abundant endocrine-disrupting compound that is found in the aquatic environment and has adverse effects on fish reproduction; however, the exact pathway of these impacts is ...unclear. In this study, the different effects of BPA on ovarian and testis development in goldfish (Carassius auratus) and the different mechanisms underlying these effects were investigated. The gonadosomatic index (GSI) and gonadal histology demonstrated that BPA diminished ovarian maturation in goldfish, which recovered after BPA treatment withdrawal. In males, BPA disrupted testis maturation, but this disruption could not be recovered after BPA treatment withdrawal. The hypothalamic-pituitary-gonad (HPG) axis-related genes sgnrh, fshβ and lhβ were significantly decreased in BPA-treated female fish, while no changes in sex steroid hormone levels and no TUNEL and PCNA staining were found in the ovary, suggesting that BPA may reduce ovarian maturation through the HPG axis. In male fish, TUNEL staining was found in 1 μg L−1 BPA-exposed germ cells and 50 and 500 μg L−1 BPA-exposed Leydig cells. Decreases in 11-KT levels were also found in 50 and 500 μg L−1 BPA-exposed fish, but BPA did not affect genes associated with the HPG axes. This result shows that BPA disrupts testis maturation through apoptosis of germ cells and Leydig cells, thus inducing decreases in 11-KT levels that disrupt spermatogenesis. Collectively, our findings provide insights into the molecular and cellular mechanisms underlying BPA disturbance of goldfish reproduction.
BPA reduced ovarian maturation by affecting the HPG axis. BPA disrupted testis maturation through apoptosis of germ and Leydig cells, reduction of 11-KT plasma levels and altered spermatogenesis. Display omitted
•BPA diminished ovarian maturation and this could be recovered after BPA treatment withdrawal.•BPA disrupted testis maturation and this disruption cannot be recovered after BPA treatment withdrawals.•BPA reduced ovarian maturation by affecting the HPG axis.•BPA disrupted testis maturation through apoptosis of germ and Leydig cells, reduce androgen levels and altered spermatogenesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
INTRODUCTION: Berberis vulgaris extract is known to be protective against many damages due to its anti-inflamatory and antioxidant properties. Paracetamol toxicity is one of the most common ...toxicities. In our study, we aimed to reduce the damage caused by paracetamol toxicity with the beneficial effects of Berberis vullgaris extract. METHODS: In our study, 42 Sprague Dawley 8-week-old rats were used. 7 groups were formed with 6 female animals in each group. At the 24th hour of the study, all groups underwent laparotomy under anesthesia and liver dissection was performed. Hematoxylin and Eosin (H&E) staining was used to evaluate liver histopathology, and PAS staining was used to show glycogen and basement membrane staining in liver tissue. In addition, scoring was done by showing apoptotic cells with TUNEL stain RESULTS: Hepatic degeneration and peritubular inflammation were quite evident in the groups to which we applied paracetamol. In this study, we observed that the number of positive cells increased in the group given paracetamol in TUNEL staining. As a result of the PAS staining we performed, glycogen accumulation in the hepatocytes of the group we induced with paracetamol was remarkable. DISCUSSION AND CONCLUSION: Berberis vulgaris plant extract appears to reduce Paracetamol-induced hepatic toxicity. As a result of the PAS staining, glycogen accumulation in the hepatocytes was remarkable in the group which was induced with paracetamol. Further studies are required for the use of determined molecules of Berberis vulgaris plant extract against the hepatotoxic effect of paracetamol in paracetamol toxicity.
Detection of merely apoptosis does not reveal the type of central nervous system (CNS) cells that are dying in the CNS diseases and injuries. In situ detection and estimation of amount of apoptosis ...specifically in neurons or glial cells (astrocytes, oligodendrocytes, and microglia) can unveil valuable information for designing therapeutics for protection of the CNS cells and functional recovery. A method was first developed and reported from our laboratory for in situ detection and estimation of amount of apoptosis precisely in neurons and glial cells using in vitro and in vivo models of CNS diseases and injuries. This is a combination of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and double immunofluorescent labeling (DIFL) or simply TUNEL-n-DIFL method for in situ detection and estimation of amount of apoptosis in a specific CNS cell type. An anti-digoxigenin (DIG) IgG antibody conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) for blue fluorescence, fluorescein isothiocyanate (FITC) for green fluorescence, or Texas Red (TR) for red fluorescence can be used for in situ detection of apoptotic cell DNA, which is earlier labeled with TUNEL using alkali-stable DIG-11-dUTP. A primary anti-NeuN (neurons), anti-GFAP (astrocytes), anti-MBP (oligodendrocytes), or anti-OX-42 (microglia) IgG antibody and a secondary IgG antibody conjugated with one of the above fluorophores (other than that of ani-DIG antibody) are used for in situ detection of apoptosis in a specific CNS cell type in the mixed culture and animal models of the CNS diseases and injuries.
Gold nanoparticles (AuNPs) have been widely used in many applications. Their usage as drug delivery vehicles has also gained considerable attention due to their chemical and optical properties as ...well as their good biocompatibility. The present study was conducted to evaluate the efficiency of AuNPs in enhancing the cytotoxic and apoptotic induction activity of lantadene A (LA), separated from Lantana camara leaves, on the breast tumor cell line MCF-7 in vitro. By utilizing plant-mediated synthesis method of nanostructures, LA-loaded AuNPs (LA-AuNPs) were prepared and their formation was confirmed by means of ultraviolet–visible spectroscope, atomic force microscope, scanning electron microscope, and zeta potential. The cytotoxic effect of LA-AuNPs was analyzed using a methylthiazol tetrazolium assay and compared to free AuNPs and LA. The results indicated a significant increase in the reduction of MCF-7 cells viability after incubation with LA-AuNPs. As determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, LA-AuNPs induced a greater ratio of DNA-fragmented cells compared to LA-treated and untreated cells. Also, by operating real-time polymerase chain reaction, LA-AuNPs-treated cells displayed an increased upregulation of p53 expression and downregulation of BCL-2 expression in addition to a significant reduction in the level of BCL-2-BAX ratio. No significant effect was shown on the expression of BAX. Collectively, our results indicate that LA-AuNPs showed promising cytotoxicity to MCF-7 cells as a novel nanoscale preparation, likely via induction of apoptotic genes and stimulation of DNA fragmentation.
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NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
This study has aimed to investigate the relationship between salt stress, programmed cell death (PCD) and microtubule distribution in terms of duration and stress dose. PCD is an important mechanism ...that benefits living organisms throughout their lives. On the other hand, PCD is an indirect effect that reduces efficiency when it occurs under stress. In this research The maize (Zea mays) roots were exposed to salt stress with 0, 50, 100, 300 and 500 mM NaCl. The prepared paraffin sections of these five groups were subjected to DAPI (4-6-diamidino-2-phenylindole) and TUNEL analysis to study the morphological changes caused by stress-induced nuclear degeneration. PCD was determined. Microtubule labeling analysis was performed on the tissues to determine whether there were stress-induced microtubule changes in these cells and disturbances were found; they exhibited aggregation, regional thickening, and random distribution around the nucleus and vacuole and under the cell wall. When all groups were evaluated, cells exposed to a salt concentration of 50 mM (even after 24 hours) were significantly less damaged than cells at other concentrations (100, 300, and 500 mM) at each time point. The rate of progression and spread to the whole tissue was significantly higher at 300 and 500 mM salt concentrations compared to the other groups. To reduce economic losses in salty soils, it is of great importance to fully investigate stress. The data that will emerge from our research, which is the subject of a small number of studies, will help to understand the mechanism of stress, microtubule and PCD.
Chemotherapy using cytotoxic agents, such as letrozole (LTZ), is one of the most effective treatments for hormone-dependent breast cancer. Nevertheless, nonspecific targeting of the drug constructs ...several remarkable systemic toxicities. In this study, we synthesized solid lipid nanoparticles (SLNs) by solvent emulsification evaporation method as LTZ carriers. Nanoparticles were also modified with a cancer cell-targeting ligand, folic acid (FA), and then characterized. Cell membrane damage and cell viability were investigated by lactate dehydrogenase (LDH) and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays, respectively. Caspase-3 activity and TUNEL assays were performed to verify induced apoptosis. Scanning electron microscopy (SEM) exhibited uniform and spherical morphology of the SLNs-LTZ and FA-SLNs-LTZ. The X-ray diffraction (XRD) confirmed LTZ was dispersed as amorphous in the SLNs. The cell culture results revealed that FA-SLNs-LTZ was significantly more cytotoxic than SLNs-LTZ and free drug against MCF-7 cancer cells in vitro, with a 50% inhibitory concentration (IC50) value of 81 ± 0.89 nM, but both nanoformulations had negligible cytotoxicity toward MCF-10A normal cells and they showed promising biocompatibility. Taken together, these findings indicated the evidence of apoptosis as a mechanism of cell death. This study suggests the potential of FA-SLNs-LTZ for inducing apoptosis in a target-specific manner with minimal systemic side effects.
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IJS, IZUM, KILJ, NUK, PILJ, SAZU, UL, UM, UPUK
Several clinical laboratories assess sperm DNA fragmentation (sDF) in addition to semen analysis in male infertility diagnosis. Among tests evaluating sDF, TUNEL (Terminal deoxynucleotidyl ...transferase dUTP nick end labeling) and SCD (Sperm Chromatin Dispersion) are widely used. Our lab developed a modified version of TUNEL (TUNEL/PI) able to distinguish two sperm populations (PI Brighter and PI Dimmer) differently associated with sperm viability and reproductive outcomes. The aim of this study was to compare sDF levels detected by SCD and TUNEL/PI in the semen samples from 71 male subjects attending our Andrology Laboratory. Our results demonstrate that SCD is less sensitive in determining sDF compared to TUNEL/PI. The statistically significant positive correlation found between sDF evaluated by SCD and PI Dimmer (consisting of all dead spermatozoa) suggests that SCD mainly detects sDF in unviable spermatozoa. We confirmed that most spermatozoa detected by SCD are unviable by performing SCD after incubation in hypo-osmotic medium to discriminate viable and unviable cells in 52 samples. Such results might explain the lower ability of this test in discriminating couples having successful ART outcomes demonstrated in published metanalyses. Overall, our results indicate that SCD is less sensitive in evaluating sDF for diagnostic purposes.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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