• Title, Summary, Keyword: DNA fragmentation

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Protective Effects of Chitosan on the Cadmium Cytotoxicity in Rat Glioma Cells (흰쥐 신경교종세포에서 카드뮴 세포독성에 대한 키토산의 효과)

  • 백용아;이정래;김강득;김혜원;이한솔;허정무;오재민;최민규;정연태
    • Toxicological Research
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    • v.20 no.1
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    • pp.63-69
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    • 2004
  • Casapse-3 protease is known as a key role of apoptotic enzyme, and caspase-3 activity is a central event that occurs upstream of DNA fragmentation during apoptosis. This study demonstrates that chitosan pretreatment inhibits cadmium-induced apoptosis by attenuating the activity of caspase-3. We also analyzed the protective effect of chitosan on DNA fragmentation induced by cadmium. Cadmium toxicity was examined by DNA fragmentation and nuclear condensation with Hoechst stain. Caspase-3 activities were increased cadmium treated group for 3 hours compared with control. When chitosan (150 mg/ml) was pretreated at 30 min before cadmium treatment, cadmium cytotoxicity was suppressed in a dose-dependent manner evaluated by DNA fragmentation and caspase activity. From these results, it is suggest that the protective effect of chitosan pretreatment against cadmium-induced cytotoxicity is mediated through inhibition of caspase-3 protease activation and DNA fragmentation.

Evaluation of normal morphology, DNA fragmentation, and hyaluronic acid binding ability of human spermatozoa after using four different commercial media for density gradient centrifugation

  • Lee, Dayong;Jee, Byung Chul
    • Clinical and Experimental Reproductive Medicine
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    • v.46 no.1
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    • pp.8-13
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    • 2019
  • Objective: Density gradient centrifugation (DGC) is frequently used to isolate high-motility fractions of spermatozoa. We compared the efficacy of four DGC media in terms of the percentage of morphologically normal spermatozoa, DNA fragmentation level, and hyaluronic acid (HA) binding ability. Methods: Thirty men with a total motile spermatozoa count > 80 million participated. Semen samples were divided into four aliquots, which were processed using PureSperm, PureCeption, Sidney, and SpermGrad media, respectively. The DNA fragmentation level was measured using the Halosperm assay kit and HA binding ability was measured using the HBA assay kit. Results: The mean percentage of morphologically normal spermatozoa was significantly enhanced after DGC using all four media (10.3%, 9.9%, 9.8%, and 10.7%, respectively; p< 0.05 for each when compared with 6.9% in raw semen). The DNA fragmentation level was significantly reduced after DGC using PureSperm, PureCeption, and SpermGrad media (6.0%, 6.5%, and 4.9%, respectively; p< 0.05 for each when compared with 11.2% in raw semen), but not after DGC using Sidney media (8.5%, p> 0.05). HA binding ability did not change after DGC using any of the four media. Conclusion: The four media were equally effective for obtaining a sperm fraction with highly motile, morphologically normal sperm. PureSperm, PureCeption, and SpermGrad media were equally effective for acquiring a sperm fraction with less DNA fragmentation.

Pretreatment of Low Dose Radiation Reduces Radiation-Induced Apoptosis in Mouse Lymphoma (EL4) cells

  • Kim, Jeong-Hee;Hyun, Soo-Jin;Yoon, Moon-Young;Jioon, Young-Hoon;Cho, Chul-Koo;Yoo, Seong-Yul
    • Archives of Pharmacal Research
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    • v.20 no.3
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    • pp.212-217
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    • 1997
  • Induction of an adaptive response to ionizing radiation in mouse lymphoma (EL4) cells was studied by using cell survival fraction and apoptotic nucleosomal DNA fragmentation as biological end points. Cells in early log phase were pre-exposed to low dose of ${\gamma}$-rays (0.01 Gy) 4 or 20 hrs prior to high dose ${\gamma}$-ray (4, 8 and 12 Gy for cell survival fraction analysis; 8 Gy for DNA fragmentation analysis) irradiation. Then cell survival fractions and the extent of DNA fragmentation were measured. Significant adaptive response, increase in cell survival fraction and decrease in the extent of DNA fragmentation were induced when low and high dose .gamma.-ray irradiation time interval was 4 hr. Addition of protein or RNA synthesis inhibitor, cycloheximide or 5,6-dichloro-1-.betha.-d-ribofuranosylbenzimidazole (DRFB), respectively during adaptation period, the period from low dose ${\gamma}$-ray irradiation to high dose ${\gamma}$-ray irradiation, was able to inhibit the induction of adaptive response, which is the reduction of the extent DNA fragmentation in irradiated EL4 cells. These data suggest that the induction of adaptive response to ionizing radiation in EL4 cells required both protein and RNA synthesis.

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Induction of Apoptosis by Camptothecin in HL-60 Cells (HL-60 세포에서 Camptothecin의 apoptosis 유도작용)

  • 김해종;천영진;김미영
    • YAKHAK HOEJI
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    • v.43 no.3
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    • pp.385-390
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    • 1999
  • Camptothecin (CPT) has been known to induce apoptosis in various cancer cell lines. To examine the intracellular apoptotic death signal initiated by CPT, we investigated the possible connection between caspase-3 activation and GSH depletion during CPT-induced apoptosis in HL-60 cells. Treatment of cells with $1{\;}{\mu}M$ CPT induced PARP cleavage accompanied by DNA fragmentation. z-VAD-fmk, a caspase-3 inhibitor, blocked the CPT-induced DNA fragmentation. Pretreatment of cells with N-acetylcysteine, a precursor of GSH biosynthesis, failed to inhibit CPT-induced PARP celavage and DNA gragmenatation. No significant changes in GSH depletion is not essential for caspase activation during CPT-induced apoptosis. We also investigated whether CPT-induced apoptosis is associated with changes of the levels of Bax and Bcl-2, two proteins involved in the control of apoptosis. Bcl-2 levels exhibited a late decrease compared with the kinetics of DNA fragmentation, whereas Bax levels increased more rapidly after CPT treatment. These results suggest that Bax plays more important role than Bcl-2 in inducing DNA fragmentation and may function upsteam of proteolytic activation of caspase-3 pathway in CPT-induced apoptosis.

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A New Bicistronic Fragmentation Vector for Manipulation and Analysis of Functional Yeast Artificial Chromosomes (YACs) (Yeast Artificial Chromosome의 효율적인 조작과 분석을 위한 새로운 Bicistronic Fragmentation Vector의 개발에 관한 연구)

  • 임향숙;최주연;김인경;강성만;성영모
    • Korean Journal of Microbiology
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    • v.35 no.1
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    • pp.28-34
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    • 1999
  • Fragmentation vectors are used to analyze function and genomic structure of a gene of interest by creating deletion derivatives of large fragments of genomic DNA cloned as yeast artificial chromosomes (YACs). Herein, we developed a new hicistronic fragmentation vector that contains internal ribosomal entry sile (IRES) of encephalomyocarditis vin~s (EMCV) and $\beta$-galactosidase as a reporter gene. This vector system provides a novcl loo1 to analyze expression patterns of a gene of interest due to simultaneous expression of a target gene as well as $\beta$-galactosidase driven from a single message. In addition, the bicistronic fragmentation vector contains four rare-cutting restriction enzyme sites in the polycloning sites which can be used to conveniently insert any kinds of genes and therefore facilitates targeting DNA scgments into YAC by means of homologous recombination. This approach establishes a paradigm for manipulation of mammalian DNA segments and characterization of expression and regulatory regions of mammalian gene cloned as YAC.

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What should be done for men with sperm DNA fragmentation?

  • Kim, Gi Young
    • Clinical and Experimental Reproductive Medicine
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    • v.45 no.3
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    • pp.101-109
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    • 2018
  • In an age when a small quantity of sperm can lead to pregnancy through in vitro fertilization or intracytoplasmic sperm injection, selecting healthy sperm is important. Sperm DNA fragmentation (SDF) is known to be higher in infertile men. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and the alkaline comet test are SDF tests that directly measure DNA damage and have shown closer correlations with assisted reproduction results than indirect tools such as the sperm chromatin structure assay or the sperm chromatic dispersion test. It is difficult; however, to endorse a single test as the best test overall; instead, it is best to select a testing method based on each patient's clinical condition and goals. In a couple struggling with infertility, if the male partner has a high level of SDF, he should aim to decrease SDF through lifestyle modifications, antioxidant treatment, and ensuring an appropriate duration of abstinence, and physicians need to treat the underlying diseases of such patients. If sperm DNA damage continues despite the patient's and physician's efforts, other methods, such as micromanipulation-based sperm selection or testicular sperm extraction, should be used to select healthy sperm with nuclear DNA integrity.

Efficient isolation of sperm with high DNA integrity and stable chromatin packaging by a combination of density-gradient centrifugation and magnetic-activated cell sorting

  • Chi, Hee-Jun;Kwak, Su-Jin;Kim, Seok-Gi;Kim, Youn-Young;Park, Ji-Young;Yoo, Chang-Seok;Park, Il-Hae;Sun, Hong-Gil;Kim, Jae-Won;Lee, Kyeong-Ho
    • Clinical and Experimental Reproductive Medicine
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    • v.43 no.4
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    • pp.199-206
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    • 2016
  • Objective: This study was carried out to investigate the correlations of the sperm DNA fragmentation index (DFI) with semen parameters and apoptosis, and to investigate the effects of density-gradient centrifugation (DGC) and magnetic-activated cell sorting (MACS) on reducing the proportion of sperm with DNA fragmentation and protamine deficiency. Methods: Semen analysis and a sperm DNA fragmentation assay were performed to assess the correlations between semen parameters and the DFI in 458 semen samples. Sperm with progressive motility or non-apoptosis were isolated by DGC or MACS, respectively, in 29 normozoospermic semen samples. The effects of DGC or MACS alone and of DGC and MACS combined on reducing the amount of sperm in the sample with DNA fragmentation and protamine deficiency were investigated. Results: The sperm DFI showed a significant correlation (r=-0.347, p< 0.001) with sperm motility and morphology (r=-0.114, p< 0.05) but not with other semen parameters. The DFI ($11.5%{\pm}2.0%$) of semen samples was significantly reduced by DGC ($8.1%{\pm}4.1%$) or MACS alone ($7.4%{\pm}3.9%$) (p< 0.05). The DFI was significantly further reduced by a combination of DGC and MACS ($4.1%{\pm}1.3%$, p< 0.05). Moreover, the combination of DGC and MACS ($1.6%{\pm}1.1%$, p< 0.05) significantly reduced the protamine deficiency rate of semen samples compared to DGC ($4.4%{\pm}3.2%$) or MACS alone ($3.4%{\pm}2.2%$). Conclusion: The combination of DGC and MACS may be an effective method to isolate high-quality sperm with progressive motility, non-apoptosis, high DNA integrity, and low protamine deficiency in clinical use.

Studies on the Regulation of Ovarian Granulosa Cell Apoptosis by Gonadotropins and Nitric Oxide (생식소 자극 호르몬과 Nitric Oxide에 의한 난소 과립세포의 Apoptosis 조절에 대한 연구)

  • 이석자
    • Development and Reproduction
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    • v.1 no.2
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    • pp.157-164
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    • 1997
  • To study the regulation of porcine follicular cell apostosis by gonadotropin, steroid, and nitric oxide, we analyzed DNA fragmentation, the hallmark of apoptosis, and nitrite production of porcine granulosa cells. Dissected indiidual follicles from ovary were separated in size (small, 2-3 mm; medium, 5-6 mm; large, 7-8 mm) and isolated granulosa cells were classified morpholocally as atretic or nonatretic. Nitrite concentration was measured by mixing follicular fluids with an equal volume of Griess reagent. Follicular nitric oxide (NO) concentration of healthy follicles was higher than that of atretic follicles. Apoptotic DNA fragmentation was suppressed in non-apoptotic granulosa cells. Follicular apoptosis was induced by androgen but prevented by gonadotropin in vitro. Apoptosis was confined to the granulosa cells. But it was not clear whether apoptosis of granulosa cells were isolated, incubated with or without gonadotropin, androgen and sodium nitroprusside (SNP), respectively at $37^{\circ}C$ for 24 hrs. Cultured granulosa cells were used to extract genomic DNA and culture media was asssayed for nitrite concentration. Nitrite production of culture media was increased, while apoptotic DNA fragmentation was suppressed in PMSG, hCG, testosterone+SNP and SNP treated groups. Nitrite concentration in culture media was decreased, but apoptotic DNA fragmentation was induced in testosterone treated group. These data suggest that NO production and apoptosis may be involved of granulosa cell apoptosis induced by testosterone.

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Seminal Tumor Necrosis Factor-alpha Level and Sperm Nuclear DNA Integrity in Healthy Donors (정액 내 Tumor Necrosis Factor-alpha 농도와 정자 DNA 손상과의 관련성)

  • Kim, Hyun-Jun;Jee, Byung-Chul;Moon, Jeong-Hee;Lee, Jung-Ryeol;Suh, Chang-Suk;Kim, Seok-Hyun
    • Clinical and Experimental Reproductive Medicine
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    • v.36 no.1
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    • pp.35-43
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    • 2009
  • Objectives: Seminal concentration of tumor necrosis factor-alpha (TNF-${\alpha}$) relevant to sperm nuclear DNA integrity has not been studied. The present study aimed to evaluate seminal concentration of TNF-${\alpha}$ in correlation with sperm parameters and nuclear DNA integrity in asymptomatic healthy donors. Methods: Semen samples were obtained by masturbation from forty-five healthy donors. Results: Sperm quality was assessed by computer-assisted semen analysis and nuclear DNA integrity measured by the TUNEL assay in raw semen. TNF-${\alpha}$ concentrations were measured by ELISA in frozen-thawed seminal plasmas. Sperm DNA fragmentation rates were ranged between 1.9% and 53.0% (mean${\pm}$SD, 12.4${\pm}$9.6%). Univariate analysis revealed that DNA fragmentation rate was not associated with sperm concentration or motility but had a correlation with linearity negatively (r=-0.325, p=0.03) and age positively (r=0.484, p=0.001). The mean seminal concentration of TNF-${\alpha}$ was 4.9 pg/mL with a range from 1.1 to 22.6 pg/mL. The TNF-${\alpha}$ concentration had no correlation with clinically relevant parameters of sperm quality or nuclear DNA fragmentation rate. Conclusion: Our results indicate that sperm nuclear DNA fragmentation may be not associated with seminal TNF-${\alpha}$ level or sperm quality in asymptomatic healthy donors.

Does conventional freezing affect sperm DNA fragmentation?

  • Le, Minh Tam;Nguyen, Thai Thanh Thi;Nguyen, Tung Thanh;Nguyen, Trung Van;Nguyen, Tam An Thi;Nguyen, Quoc Huy Vu;Cao, Thanh Ngoc
    • Clinical and Experimental Reproductive Medicine
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    • v.46 no.2
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    • pp.67-75
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    • 2019
  • Objective: Sperm cryopreservation has been widely used in assisted reproductive technology, as it offers great potential for the treatment of some types of male infertility. However, cryopreservation may result in changes in membrane lipid composition and acrosome status, as well as reductions in sperm motility and viability. This study aimed to evaluate sperm DNA fragmentation damage caused by conventional freezing using the sperm chromatin dispersion test. Methods: In total, 120 fresh human semen samples were frozen by conventional methods, using SpermFreeze Solution as a cryoprotectant. Routine semen analysis and a Halosperm test (using the Halosperm kit) were performed on each sample before freezing and after thawing. Semen parameters and sperm DNA fragmentation were compared between these groups. Results: There was a significant decrease in sperm progressive motility, viability, and normal morphology after conventional freezing (32.78%, 79.58%, and 3.87% vs. 16%, 55.99%, and 2.55%, respectively). The sperm head, midpiece, and tail defect rate increased slightly after freezing. Furthermore, the DNA fragmentation index (DFI) was significantly higher after thawing than before freezing (19.21% prior to freezing vs. 22.23% after thawing). Significant increases in the DFI after cryopreservation were observed in samples with both normal and abnormal motility and morphology, as well as in those with normal viability. Conclusion: Conventional freezing seems to damage some sperm parameters, in particular causing a reduction in sperm DNA integrity.